Nuclear transport modulators and uses thereof

ABSTRACT

and pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising the compounds of formula I, and methods of using said compounds, salts and compositions in the treatment of various disorders associated with CRM1 activity.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/747,394, filed Jun. 23, 2015, which is a continuation of U.S.application Ser. No. 13/891,044, filed on May 9, 2013, which claims thebenefit of U.S. Provisional Application No. 61/798,188, filed on Mar.15, 2013 and U.S. Provisional Application No. 61/644,802, filed on May9, 2012. The entire teachings of the above applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Cells from most major human solid and hematologic malignancies exhibitabnormal cellular localization of a variety of oncogenic proteins, tumorsuppressor proteins, and cell cycle regulators (Cronshaw et al, 2004,Falini et al 2006). For example, certain p53 mutations lead tolocalization in the cytoplasm rather than in the nucleus. This resultsin the loss of normal growth regulation, despite intact tumor suppressorfunction. In other tumors, wild-type p53 is sequestered in the cytoplasmor rapidly degraded, again leading to loss of its suppressor function.Restoration of appropriate nuclear localization of functional p53protein can normalize some properties of neoplastic cells (Cai et al,2008; Hoshino et al 2008; Lain et al 1999a; Lain et al 1999b; Smart etal 1999), can restore sensitivity of cancer cells to DNA damaging agents(Cai et al, 2008), and can lead to regression of established tumors(Sharpless & DePinho 2007, Xue et al, 2007). Similar data have beenobtained for other tumor suppressor proteins such as forkhead (Turnerand Sullivan 2008) and c-Abl (Vignari and Wang 2001). In addition,abnormal localization of several tumor suppressor and growth regulatoryproteins may be involved in the pathogenesis of autoimmune diseases(Davis 2007, Nakahara 2009). CRM1 inhibition may provide particularlyinteresting utility in familial cancer syndromes (e.g., Li-FraumeniSyndrome due to loss of one p53 allele, BRCA1 or 2 cancer syndromes),where specific tumor suppressor proteins (TSP) are deleted ordysfunctional and where increasing TSP levels by systemic (or local)administration of CRM1 inhibitors could help restore normal tumorsuppressor function.

Specific proteins and RNAs are carried into and out of the nucleus byspecialized transport molecules, which are classified as importins ifthey transport molecules into the nucleus, and exportins if theytransport molecules out of the nucleus (Terry et al, 2007; Sorokin et al2007). Proteins that are transported into or out of the nucleus containnuclear import/localization (NLS) or export (NES) sequences that allowthem to interact with the relevant transporters. Chromosomal RegionMaintenance 1 (Crm1), which is also called exportin-1 or Xpol, is amajor exportin.

Overexpression of Crm1 has been reported in several tumors, includinghuman ovarian cancer (Noske et al, 2008), cervical cancer (van der Wattet al, 2009), pancreatic cancer (Huang et al, 2009), hepatocellularcarcinoma (Pascale et al, 2005) and osteosarcoma (Yao et al, 2009) andis independently correlated with poor clinical outcomes in these tumortypes.

Inhibition of Crm1 blocks the exodus of tumor suppressor proteins and/orgrowth regulators such as p53, c-Abl, p21, p27, pRB, BRCA1, IkB, ICp27,E2F4, KLF5, YAP1, ZAP, KLF5, HDAC4, HDAC5 or forkhead proteins (e.g.FOXO3a) from the nucleus that are associated with gene expression, cellproliferation, angiogenesis and epigenetics. Crm1 inhibitors have beenshown to induce apoptosis in cancer cells even in the presence ofactivating oncogenic or growth stimulating signals, while sparing normal(untransformed) cells. Most studies of Crm1 inhibition have utilized thenatural product Crm1 inhibitor Leptomycin B (LMB). LMB itself is highlytoxic to neoplastic cells, but poorly tolerated with markedgastrointestinal toxicity in animals (Roberts et al, 1986) and humans(Newlands et al, 1996). Derivatization of LMB to improve drug-likeproperties leads to compounds that retain antitumor activity and arebetter tolerated in animal tumor models (Yang et al, 2007, Yang et al,2008, Mutka et al, 2009). Therefore, nuclear export inhibitors couldhave beneficial effects in neoplastic and other proliferative disorders.To date, however, small-molecule, drug-like Crm1 inhibitors for use invitro and in vivo are uncommon.

In addition to tumor suppressor proteins, Crm1 also exports several keyproteins that are involved in many inflammatory processes. These includeIkB, NF-kB, Cox-2, RXRα, Commd1, HIF1, HMGB1, FOXO, FOXP and others. Thenuclear factor kappa B (NF-kB/rel) family of transcriptional activators,named for the discovery that it drives immunoglobulin kappa geneexpression, regulate the mRNA expression of variety of genes involved ininflammation, proliferation, immunity and cell survival. Under basalconditions, a protein inhibitor of NF-kB, called IkB, binds to NF-kB inthe nucleus and the complex IkB-NF-kB renders the NF-kB transcriptionalfunction inactive. In response to inflammatory stimuli, IkB dissociatesfrom the IkB-NF-kB complex, which releases NF-kB and unmasks its potenttranscriptional activity. Many signals that activate NF-kB do so bytargeting IkB for proteolysis (Phosphorylation of IkB renders it“marked” for ubiquitination and then proteolysis). The nuclearIkBa-NF-kB complex can be exported to the cytoplasm by Crm1 where itdissociates and NF-kB can be reactivated. Ubiquitinated IkB may alsodissociate from the NF-kB complex, restoring NF-kB transcriptionalactivity. Inhibition of Crm1 induced export in human neutrophils andmacrophage like cells (U937) by LMB not only results in accumulation oftranscriptionally inactive, nuclear IkBa-NF-kB complex but also preventsthe initial activation of NF-kB even upon cell stimulation (Ghosh 2008,Huang 2000). In a different study, treatment with LMB inhibited IL-1βinduced NF-kB DNA binding (the first step in NF-kB transcriptionalactivation), IL-8 expression and intercellular adhesion moleculeexpression in pulmonary microvascular endothelial cells (Walsh 2008).COMMD1 is another nuclear inhibitor of both NF-kB and hypoxia-induciblefactor 1 (HIF1) transcriptional activity. Blocking the nuclear export ofCOMMD1 by inhibiting Crm1 results in increased inhibition of NF-kB andHIF1 transcriptional activity (Muller 2009).

Crm1 also mediates Retinoid X receptor α (RXRα) transport. RXRα ishighly expressed in the liver and plays a central role in regulatingbile acid, cholesterol, fatty acid, steroid and xenobiotic metabolismand homeostasis. During liver inflammation, nuclear RXRα levels aresignificantly reduced, mainly due to inflammation-mediated nuclearexport of RXRα by Crm1. Lep B is able to prevent IL-1β inducedcytoplasmic increase in RXRα levels in human liver derived cells(Zimmerman 2006).

The role of Crm1-mediated nuclear export in NF-kB, HIF-1 and RXRαsignalling suggests that blocking nuclear export can be potentiallybeneficial in many inflammatory processes across multiple tissues andorgans including the vasculature (vasculitis, arteritis, polymyalgiarheumatic, atherosclerosis), dermatologic (see above), rheumatologic(rheumatoid and related arthritis, psoriatic arthritis,spondyloarthropathies, crystal arthropathies, systemic lupuserythematosus, mixed connective tissue disease, myositis syndromes,dermatomyositis, inclusion body myositis, undifferentiated connectivetissue disease, Sjogren's syndrome, scleroderma and overlap syndromes,etc.).

CRM1 Inhibition affects gene expression by inhibiting/activating aseries of transcription factors like ICp27, E2F4, KLF5, YAP1, ZAP

Crm1 inhibition has potential therapeutic effects across manydermatologic syndromes including inflammatory dermatoses (atopy,allergic dermatitis, chemical dermatitis, psoriasis), sun-damage(Ultraviolet/UV damage), and infections. CRM1 inhibition, best studiedwith LMB, showed minimal effects on normal keratinocytes, and exertedanti-inflammatory activity on keratinocytes subjected to UV, TNFa, orother inflammatory stimuli (Kobayashi & Shinkai 2005, Kannan & Jaiswal2006). Crm1 inhibition also upregulates NRF2 (nuclear factorerythroid-related factor 2) activity, which protects keratinocytes(Schafer et al, 2010, Kannan & Jaiswal 2006) and other cell types (Wanget al, 2009) from oxidative damage. LMB induces apoptosis inkeratinocytes infected with oncogenic human papillomavirus (HPV) strainssuch as HPV16, but not in uninfected keratinocytes (Jolly et al, 2009).

Crm1 also mediates the transport of key neuroprotectant proteins thatmay be useful in neurodegenerative diseases including Parkinson'sDisease (PD), Alzheimer's Disease, and Amyotrophic Lateral Sclerosis.For example, (1) forcing nuclear retention of key neuroprotectiveregulators such as NRF2 (Wang 2009), FOXA2 (Kittappa et al, 2007),parking in neuronal cells and/or by (2) inhibiting NFκB transcriptionalactivity by sequestering IκB to the nucleus in glial cells, Crm1inhibition could slow or prevent neuronal cell death found in thesedisorders. There is also evidence linking abnormal glial cellproliferation to abnormalities in CRM1 levels or CR1 function (Shen2008).

Intact nuclear export, primarily mediated through CRM1, is also requiredfor the intact maturation of many viruses. Viruses where nuclear export,and/or CRM1 itself, has been implicated in their lifecycle include humanimmunodeficiency virus (HIV), adenovirus, simian retrovirus type 1,Borna disease virus, influenza (usual strains as well as H1N1 and avianH5N1 strains), hepatitis B (HBV) and C (HCV) viruses, humanpapillomavirus (HPV), respiratory syncytial virus (RSV), Dungee, SevereAcute Respiratory Syndrome coronavirus, yellow fever virus, West NileVirus, herpes simplex virus (HSV), cytomegalovirus (CMV), and Merkelcell polyomavirus (MCV). (Bhuvanakantham 2010, Cohen 2010, Whittaker1998). It is anticipated that additional viral infections reliant onintact nuclear export will be uncovered in the near future.

The HIV-1 Rev protein, which traffics through nucleolus and shuttlesbetween the nucleus and cytoplasm, facilitates export of unspliced andsingly spliced HIV transcripts containing Rev Response Elements (RRE)RNA by the CRM1 export pathway. Inhibition of Rev-mediated RNA transportusing CRM1 inhibitors such as LepB or PKF050-638 can arrest the HIV-1transcriptional process, inhibit the production of new HIV-1 virions,and thereby reduce HIV-1 levels (Pollard 1998, Daelemans 2002).

Dengue virus (DENV) is the causative agent of the common arthropod-borneviral disease, dengue fever (DF), and its more severe and potentiallydeadly dengue hemorrhagic fever (DHF). DHF appears to be the result ofan over exuberant inflammatory response to DENV. NS5 is the largest andmost conserved protein of DENV. CRM1 regulates the transport of NS5 fromthe nucleus to the cytoplasm, where most of the NS5 functions aremediated. Inhibition of CRM1 mediated export of NS5 results in alteredkinetics of virus production and reduces induction of the inflammatorychemokine interleukin-8 (IL-8), presenting a new avenue for thetreatment of diseases caused by DENV and other medically importantflaviviruses including Hepatitis C virus (Rawlinson 2009).

Other virus-encoded RNA-binding proteins that use CRM1 to exit thenucleus include the HSV type 1 tegument protein (VP13/14, or hUL47),human CMV protein pp65, the SARS Coronavirus ORF 3b Protein, and the RSVmatrix (M) protein (Williams 2008, Sanchez 2007, Freundt 2009, Ghildyal2009).

Interestingly, many of these viruses are associated with specific typesof human cancer including hepatocellular carcinoma (HCC) due to chronicHBV or HCV infection, cervical cancer due to HPV, and Merkel cellcarcinoma associated with MCV. CRM1 inhibitors could therefore havebeneficial effects on both the viral infectious process as well as onthe process of neoplastic transformation due to these viruses.

CRM1 controls the nuclear localization and therefore activity ofmultiple DNA metabolizing enzymes including histone deacetylases (HDAC),histone acetyltransferases (HAT), and histone methyltransferases (HMT).Suppression of cardiomyocyte hypertrophy with irreversible CRM1inhibitors has been demonstrated and is believed to be linked to nuclearretention (and activation) of HDAC 5, an enzyme known to suppress ahypertrophic genetic program (Monovich et al, 2009). Thus, CRM1inhibition may have beneficial effects in hypertrophic syndromes,including certain forms of congestive heart failure and hypertrophiccardiomyopathies.

CRM1 has also been linked to other disorders. Leber's disorder, ahereditary disorder characterized by degeneration of retinal ganglioncells and visual loss, is associated with inaction of the CRM1 switch(Gupta N 2008). There is also evidence linking neurodegenerativedisorders to abnormalities in nuclear transport.

In view of the above, the discovery of compounds that modulate nucleartransport is desirable.

SUMMARY OF THE INVENTION

The present invention relates to compounds, and pharmaceuticallyacceptable salts thereof, useful as nuclear transport modulators,pharmaceutically acceptable compositions comprising compounds of thepresent invention and methods of using said compositions in thetreatment of various disorders. It has now been found that nucleartransport modulators of the present invention, and pharmaceuticallyacceptable salts and/or compositions thereof, provide desirable in vivoexposure as measured by AUC in mouse while exhibiting lower levels ofbrain penetration as compared to other modulators. The compounds of theinvention have the general formula I:

or a pharmaceutically acceptable salt thereof, wherein each variable isas defined and described herein.

Compounds of the present invention and pharmaceutically acceptablecompositions thereof are useful for treating a variety of diseases,disorders or conditions, associated with abnormal cellular responsestriggered by improper nuclear transport. Such diseases, disorders, orconditions include those described herein.

Compounds provided by this invention are also useful for the study ofnuclear transport modulation in biological and pathological phenomena;the study of intracellular signal transduction pathways mediated by suchkinases; and the comparative evaluation of new nuclear transportmodulators.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention.

FIG. 1 is a graph of mean tumor volume versus time, and shows the groupmean volume of Z-138 xenograft tumors on mice treated with vehicle, 80mg/kg cyclophosphamide, 15 mg/kg Compound 2 or 7.5 mg/kg Compound 2(error bars represent SEM for each group).

FIG. 2 is a graph of mean tumor volume versus time, and shows the groupmean volume of A549 xenograft tumors on mice treated with vehicle, 5mg/kg cisplatin, 10 mg/kg Compound 2 or 5 mg/kg Compound 2 (error barsrepresent SEM for each group).

FIG. 3A is a graph of total arthritis score versus time, and shows theclinical arthritis score anti-collagen antibody induced male BALB/carthritis mice treated with vehicle, dexamethasone, 4 mg/kg Compound 2or 7.5 mg/kg Compound 2 over a 12-day observation period(¥=dexamethasone-treated group significantly different fromvehicle-treated group; #=7.5 mg/kg Compound 2-treated groupsignificantly different from vehicle-treated group; †=4 mg/kg Compound2-treated group significantly different from vehicle-treated group).

FIG. 3B is a graph of mean rear paw versus time, and shows the groupmean rear paw thickness for anti-collagen antibody induced male BALB/carthritis mice treated with vehicle, dexamethasone, 4 mg/kg Compound 2or 7.5 mg/kg Compound 2 over a 12-day observation period(¥=dexamethasone-treated group significantly different fromvehicle-treated group; #=7.5 mg/kg Compound 2-treated groupsignificantly different from vehicle-treated group; †=4 mg/kg Compound2-treated group significantly different from vehicle-treated group).

FIG. 4A is a graph of joint swelling versus time, and shows the jointswelling measured on a scale of 0-4 in naïve rats and rats treatedaccording to the CIA model, with positive control, or with Compound 2.

FIG. 4B is a graph of clinical scores as a function of time, and showsthe clinical arthritis scores of naïve rats and rats treated accordingto the CIA model, with positive control, or with Compound 2.

FIG. 5 is representative images from each treatment group in the CIAmodel, and shows the histopathology of hind paws of naïve rats and ratstreated according to the model, with positive control, or with Compound2.

FIG. 6A is a graph of ear thickness versus time, and shows the groupmean left ear thickness of female BALB/c mice treated with vehicle, PMAand vehicle, PMA and Compound 2 or PMA and betamethasone.

FIG. 6B is a graph of ear thickness versus time, and shows the groupmean right ear thickness of female BALB/c mice treated with vehicle, PMAand vehicle, PMA and Compound 2 or PMA and betamethasone.

FIG. 6C is a graph of disease activity versus time, and shows the groupmean left ear disease activity of female BALB/c mice treated withvehicle, PMA and vehicle, PMA and Compound 2 or PMA and betamethasone.

FIG. 6D is a graph of disease activity versus time, and shows the groupmean right ear disease activity of female BALB/c mice treated withvehicle, PMA and vehicle, PMA and Compound 2 or PMA and betamethasone.

FIG. 7A is a graph of disease activity index versus time, and shows thedisease activity of male BALB/c mice treated with vehicle, IMQ andvehicle, IMQ and 1 μM Compound 2, or IMQ and 10 mg/kg cyclophosphamidebefore IMQ administration.

FIG. 7B is a graph of disease activity index versus time, and shows thedisease activity of male BALB/c mice treated with vehicle, IMQ andvehicle, IMQ and 1 μM Compound 2, or IMQ and 10 mg/kg cyclophosphamideafter IMQ administration.

FIG. 8A is a graph of cumulative food intake versus time, and shows thecumulative food intake of lean Zucker rats and obese Zucker rats treatedwith vehicle (VEH), 1.5 mg/kg Compound or 3.0 mg/kg Compound 2.

FIG. 8B is a graph of average food intake versus time, and shows theaverage food intake of lean Zucker rats and obese Zucker rats treatedwith vehicle (VEH), 1.5 mg/kg Compound or 3.0 mg/kg Compound 2.

FIG. 9 is a bar graph of percentage body weight change versus time, andshows the percentage body weight change of lean Zucker rats and obeseZucker rats treated with vehicle (VEH), 1.5 mg/kg Compound or 3.0 mg/kgCompound 2 during the treatment period (Study Days 10 and 17) and duringthe washout period (Study Day 24) of the experiment.

FIG. 10A is a graph of cumulative food intake versus time, and shows thecumulative food intake of rats fed normal chow and rats fed a high-fatdiet and treated with vehicle, 1.5 mg/kg Compound or 3.0 mg/kg Compound2 during the baseline, treatment and washout phases of the study.

FIG. 10B is a graph of average body weight versus time, and shows theaverage body weight of rats fed normal chow and rats fed a high-fat dietand treated with vehicle, 1.5 mg/kg Compound or 3.0 mg/kg Compound 2during the baseline, treatment and washout phases of the study.

FIG. 11 is a bar graph of percentage body weight change versus time, andshows the percentage body weight change of rats fed normal chow and ratsfed a high-fat diet and treated with vehicle, 1.5 mg/kg Compound or 3.0mg/kg Compound 2.

FIG. 12A is a graph of Nrf2 expression under a variety of conditions,including knock-down conditions.

FIG. 12B is a graph of NQO1 expression under a variety of conditions,including knock-down conditions.

FIG. 12C is a graph of EPHX1 expression under a variety of conditions,including knowk-down conditions.

FIG. 13A is a bar graph of fold change of COX-2 mRNA expression, andshows that Compound 1 does not affect COX-2 transcription. COX-2 mRNAexpression analysis by qRT-PCR of untreated HeLa cells (control) wascompared to HeLa cells treated with 10 μM Compound 1, 20 ng/ml TNFα, or10 μM Compound 1+20 ng/ml TNFα.

FIG. 13B is a graph of intensity of COX-2 protein expression, and showsthat Compound 1 inhibits TNFα-induced COX-2 protein expression.

FIG. 14 is an image of cells treated with DMSO, 20 ng/mL TNFα, orCompound 1+20 ng/mL TNFα, and shows the localization of a variety ofinflammation-related CRM1 cargo proteins.

FIG. 15A is a graph of latency to reach platform in the MWM test as afunction of time, and shows the effect of sham treatment, controltreatment, progesterone treatment and varying doses of Compound 1 on thelatency of mice to reach the platform during the acquisition phase ofthe MWM test (data represent mean±SEM).

FIG. 15B is a graph of cytokine concentration, and shows theconcentration of several cytokines in rat plasma.

FIG. 15C is photographs of whole brains of animals receiving shamlesions (Sham), CCI+vehicle (Control), or CCI+Compound 1 (6 mg/kg), andshows the results of a qualitative visual inspection of whole brainsprior to vibratome sectioning. The inspection indicated that none (0 of4) of the Sham animals exhibited damage to dorsal-medial corticaltissue. In stark contrast, all four of the CCI controls exhibited severebilateral injury restricted to this region of the cortex. CCI animalswhich received Compound 1 showed damage ranging from moderate tominimal. Notably, the brain demonstrating the most severe injury in theCompound 1 group was less dramatic than all brains in the CCI controlgroup.

FIG. 15D is a low-power micrograph of NeuN labeling of the dorsalcortical zone and the ventral cortical zone of sham-treated (Sham),CCI+vehicle-treated (Control), and CCI+Compound 1-treated (KPT) animals.

FIG. 15E is photomicrographs of immunofluorescent labeling of Rat IgGand TNFα in sham-treated (Sham), CCI+vehicle-treated (Control), andCCI+Compound 1-treated (KPT) animals.

FIG. 16A is a graph of clinical score as a function of time, and showsthe clinical arthritis scores of naïve female Lewis rats, control femalearthritic Lewis rats, or female arthritic Lewis rats treated withCompound 2.

FIG. 16B is a graph of joint swelling as a function of time, and showsthe joint swelling measured on a scale of 0-4 in naïve female Lewisrats, control female arthritic Lewis rats, or female arthritic Lewisrats treated with Compound 2.

FIG. 17A is a graph of bone mineral density (BMD) of tarsal bones ofnaïve female Lewis rats, control female arthritic Lewis rats, and femalearthritic Lewis rats treated with Compound 2.

FIG. 17B is a visualization by three-dimensional micro CT imaging ofhind paws of naïve female Lewis rats, control female arthritic Lewisrats, and female arthritic Lewis rats treated with Compound 2.

FIG. 18A is a schematic of the MOG-induced EAE murine model in femalemice described herein.

FIG. 18B is a graph of clinical score as a function of study day, andshows the effects of vehicle treatment, dexamethasone treatment andCompound 1 treatment on the clinical score of female mice in theMOG-induced EAE murine model described herein.

FIG. 19 is photographs of wounds treated topically or systemically withCompound 1 or its appropriate vehicle, and shows the results of a woundmorphology assessment conducted on Day 5 post-wounding.

DETAILED DESCRIPTION OF THE INVENTION Compounds of the Invention

A first embodiment provides a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from hydrogen and C₁-C₄ alkyl;

R² is selected from O and S; and

R³ is selected from —N(R⁴)—(C₃-C₆ cycloalkyl), —C₁-C₆ alkyl, —(C₀-C₄alkylene)-heterocyclyl, and —(C₀-C₄ alkylene)-heteroaryl, wherein anyalkyl, alkylene, heterocyclyl, or heteroaryl portion of R³ is optionallyand independently substituted; and

R⁴ is selected from hydrogen and C₁-C₄ alkyl.

In a first aspect of the first embodiment, R¹ is selected from hydrogenand methyl. The values for the remaining variables are as described inthe first embodiment.

In a second aspect of the first embodiment, R¹ is hydrogen. The valuesfor the remaining variables are as described in the first embodiment.

In a third aspect of the first embodiment, R² is O. The values for theremaining variables are as described in the first embodiment, or firstor second aspect thereof.

In a fourth aspect of the first embodiment, R² is S. The values for theremaining variables are as described in the first embodiment, or firstthrough third aspects thereof.

In a fifth aspect of the first embodiment, R⁴ is hydrogen.

In a sixth aspect of the first embodiment, R³ is selected from—N(R⁴)—(C₃-C₆ cycloalkyl), —C₃-C₆ alkyl, —(C₀-C₁ alkylene)-heterocyclyl,and —(C₀-C₁ alkylene)-heteroaryl, wherein any alkyl or alkylene portionof R³ is optionally substituted with —N(R⁵)₂, wherein each R⁵ isindependently selected from hydrogen and C₁-C₄ alkyl; any heterocyclyl,and heteroaryl portion of R³ comprises at least one nitrogen atom in aring; and any heterocyclyl, and heteroaryl portion of R³ is optionallysubstituted with C₁-C₄ alkyl. The values for the remaining variables areas described in the first embodiment, or first through fifth aspectsthereof.

In a seventh aspect of the first embodiment, R³ is selected from—C(CH₃)₃, —CH(NH₂)—CH(CH₃)₂, —NH-cyclopropyl, —(CH₂)₀₋₁-pyrazinyl,piperidinyl, hydroxypiperidinyl, N-methylpiperidinyl,—CH₂-morpholin-4-yl, and methylpyrazolyl. The values for the remainingvariables are as described in the first embodiment, or first throughfifth aspects thereof.

In an eighth aspect of the first embodiment, R³ is selected from—C(CH₃)₃, —CH(NH₂)—CH(CH₃)₂, —NH-cyclopropyl, —(CH₂)₀₋₁-pyrazin-2-yl,piperidin-3-yl, —CH₂-morpholin-4-yl, and 5-methyl-1-H-pyrazol-4-yl. Thevalues for the remaining variables are as described in the firstembodiment, or first through fifth aspects thereof.

In a ninth aspect of the first embodiment, R³ is selected from —C(CH₃)₃,—NH-cyclopropyl, —CH₂-pyrazin-2-yl, -pyrazin-2-yl, —CH₂-morpholin-4-yl,and 5-methyl-1-H-pyrazol-4-yl. The values for the remaining variablesare as described in the first embodiment, or first through fifth aspectsthereof.

A second embodiment is a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, wherein R³ is selected from —N(R⁴)—(C₃-C₆cycloalkyl), —C₃-C₆ alkyl, —(C₀-C₁ alkylene)-heterocyclyl, and —(C₀-C₁alkylene)-heteroaryl, wherein:

any alkyl or alkylene portion of R³ is optionally substituted with—N(R⁵)₂, wherein each R⁵ is independently selected from hydrogen andC₁-C₄ alkyl;

any heterocyclyl, and heteroaryl portion of R³ comprises at least onenitrogen atom in a ring; and

any heterocyclyl, and heteroaryl portion of R³ is optionally substitutedwith C₁-C₄ alkyl.

In a first aspect of the second embodiment, R³ is selected

from —C(CH₃)₃, —CH(NH₂)—CH(CH₃)₂, —NH-cyclopropyl, —(CH₂)₀₋₁-pyrazinyl,piperidinyl, hydroxypiperidinyl, N-methylpiperidinyl,—CH₂-morpholin-4-yl, and methylpyrazolyl.

In a second aspect of the second embodiment, R³ is selected

from —C(CH₃)₃, —CH(NH₂)—CH(CH₃)₂, —NH-cyclopropyl,—(CH₂)₀₋₁-pyrazin-2-yl, piperidin-3-yl, —CH₂-morpholin-4-yl, and5-methyl-1-H-pyrazol-4-yl.

In a third aspect of the second embodiment, R³ is selected

from —C(CH₃)₃, —NH-cyclopropyl, —CH₂-pyrazin-2-yl, -pyrazin-2-yl,—CH₂-morpholin-4-yl, and 5-methyl-1-H-pyrazol-4-yl.

A third embodiment provides a compound of formula I, or apharmaceutically acceptable salt thereof, wherein R³ is selected from—N(R⁴)—(C₃-C₆ cycloalkyl), —C₃-C₆ alkyl, —(C₀-C₁ alkylene)-heterocyclyl,and —(C₀-C₁ alkylene)-heteroaryl, wherein:

any alkyl or alkylene portion of any R³ is optionally and independentlysubstituted with one or more substituents selected from the groupconsisting of oxo and —N(R⁵)₂, wherein each R⁵ is independently selectedfrom hydrogen and C₁-C₄ alkyl;

any heterocyclyl portion of R³ comprises at least one nitrogen atom in aring, and is optionally substituted with one or more substituentsselected from the group consisting of C₁-C₄ alkyl and oxo; and

any heteroaryl portion of R³ comprises at least one nitrogen atom in aring and is optionally substituted with one or more C₁-C₄ alkyl. Thevalues for the remaining variables are as described in the firstembodiment, or first through fifth aspects thereof.

In a first aspect of the third embodiment, R³ is —(C₀-C₁alkylene)-heterocyclyl. The values for the remaining variables are asdescribed in the first embodiment, or first through fifth aspectsthereof.

In a second aspect of the third embodiment, R³ is —(C₀-C₁alkylene)-heterocyclyl, wherein the heterocyclyl is selected frompyrazinyl, piperidinyl, morpholinyl, and pyrazolyl. The values for theremaining variables are as described in the first embodiment, or firstthrough fifth aspects thereof.

In a third aspect of the third embodiment, R³ is —(C₀-C₁alkylene)-heterocyclyl, wherein the heterocyclyl is morpholinyl. Thevalues for the remaining variables are as described in the firstembodiment, or first through fifth aspects thereof.

In a fourth aspect of the third embodiment, R³ is —(C₁alkylene)-heterocyclyl. The values for the remaining variables are asdescribed in the first embodiment, or first through fifth aspectsthereof.

In a fifth aspect of the third embodiment, R³ is —(C₁alkylene)-morpholinyl. The values for the remaining variables are asdescribed in the first embodiment, or first through fifth aspectsthereof.

Exemplary compounds of formula I are set forth in Table 1.

TABLE 1 Exemplary compounds of formula I. Cmpd Physical Data No.Compound Structure (¹H NMR and LCMS (M + H)⁺) 1

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.35 (s, 1H), 9.66 (s, 1H), 9.64 (s,1H), 8.57 (s, 2H), 8.28 (s, 1H), 7.48-7.50 (d, J = 8 Hz, 1H), 6.00-6.03(d, J = 12 Hz, 1H), 1.15 (s, 9H). LCMS calcd: 450.36, found: 450.19(retention time 2.89 min, purity: 94.5%). 2

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.56 (s, 1H), 9.94 (s, 1H), 9.61 (s,1H), 8.55 (s, 2H), 8.28 (s, 2H), 7.48-7.51 (d, J = 10.8 Hz, 1H),6.01-6.03 (d, J = 10.4 Hz, 1H), 3.60- 3.62 (t, 4H), 3.08 (s, 2H). LCMScalcd: 493.38, found: 493.24 (retention time 2.29 min, purity: 99.48%) 3

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 13.01 (bs, 1H), 10.47 (bs, 1H), 10.03(s, 1H), 9.70 (s, 1H), 8.56 (s, 2H), 8.28 (s, 1H), 7.97 (bs, 1H),7.51-7.54 (d, J = 10.8 Hz, 1H), 6.06- 6.08 (d, J = 10.4 Hz, 1H), 2.41(s, 3H). LCMS calcd: 474.34, found: 474.14 (retention time 2.51 min,purity: 99.88%) 4

¹H NMR (400 MHz, MeOD, ppm) δ = 9.67 (s, 1H), 8.66 (s, 2H), 8.09 (s,1H), 7.44-7.47 (d, J = 10.8 Hz, 1H), 6.02 (m, 1H), 4.64 (s, 1H), 3.33(m, 1H), 2.88 (m, 1H), 0.91 (m, 2H), 0.79 (m, 2H). LCMS calcd for:C₁₇H₁₅F₆N₆OS [M + H]⁺: 465.40, found: 465.19 (retention time 2.78 min,purity: 99.63%) 5

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.36 (s, 1H), 9.10 (s, 1H), 8.53 (s,2H), 8.29 (s, 1H), 7.32-7.34 (d, J = 10.0 Hz, 1H), 6.05- 6.07 (d, J =10.0 Hz, 1H), 3.56 (s, 4H), 3.10 (s, 3H), 3.05 (s, 2H), 2.51 (s, 4H).LCMS calcd for: C₂₀H₂₁F₆N₆O₃ [M + H]⁺: 507.41, found: 507.24 (retentiontime 2.40 min, purity: 99.61%) 6

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 9.73 (s, 1H), 8.56 (s, 2H), 8.29 (s,1H), 7.41- 7.44 (d, J = 10.4 Hz, 1H), 5.98-6.00 (d, J = 10.4 Hz, 1H),2.91 (d, 1H), 2.81 (d, 1H), 2.34-2.58 (m, 4H), 1.77 (m, 1H), 1.54 (m,2H), 1.34 (m, 1H), 1.23 (s, 2H). LCMS calcd: 477.38, found: 477.24(retention time 2.38 min, purity: 95.46%). 7

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.88 (s, 1H), 10.76 (s, 1H), 9.58(s, 1H), 8.55 (s, 2H), 8.31 (s, 1H), 8.24 (s, 2H), 7.53- 7.56 (d, J =10.4 Hz, 1H), 6.06-6.09 (d, J = 10.4 Hz, 1H), 3.69 (s, 1H), 2.13 (m,1H), 1.01 (d, 6H). LCMS calcd: 465.37, found: 465.24 (retention time2.45 min, purity: 95.19%) 8

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.95 (s, 1H), 10.82 (s, 1H), 9.62(s, 1H), 9.22 (s, 1H), 8.95 (s, 1H), 8.81 (s, 1H), 8.55 (s, 2H), 8.29(s, 1H), 7.56-7.53 (d, J = 10.4 Hz, 1H), 6.10-6.08 (d, J = 10.4 Hz, 1H).LCMS calcd: 472.32, found: 472.14 (retention time 2.68 min, purity:93.5%) 9

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.61 (s, 1H), 10.26 (s, 1H), 9.62(s, 1H), 8.57 (s, 2H), 8.30 (s, 1H), 7.52-7.49 (d, J = 10.4 Hz, 1H),6.02-6.05 (d, J = 10.4 Hz, 1H), 3.38 (m, 3H), 2.91 (m, 2H), 2.70 (s,3H), 1.80 (m, 2H), 1.76 (m, 2H). LCMS calcd: 491.41, found: 491.24(retention time 2.28 min, purity: 99.97%) 10

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.88 (s, 1H), 10.76 (s, 1H), 9.58(s, 1H), 8.55 (s, 2H), 8.31 (s, 1H), 8.24 (s, 2H), 7.53- 7.56 (d, J =10.4 Hz, 1H), 6.06-6.09 (d, J = 10.4 Hz, 1H), 3.69 (s, 1H), 2.13 (m,1H), 1.01 (d, 6H). LCMS calcd: 465.37, found: 465.24 (retention time2.45 min, purity: 95.19%) 11

¹H NMR (400 MHz, DMSO-d6, ppm) δ = 10.69 (s, 1H), 10.54 (s, 1H), 9.62(s, 1H), 8.67 (s, 1H), 8.58 (m, 2H), 8.56 (s, 2H), 8.28 (s, 1H),7.49-7.51 (d, J = 10.8 Hz, 1H), 6.01- 6.04 (d, J = 10.4 Hz, 1H), 3.84(s, 2H). LCMS calcd: 486.35, found: 486.29 (retention time 2.49 min,purity: 99.96%) 12

¹H NMR (400 MHz, DMSO-d6) δ 10.70-10.88 (m, 2H), 9.56 (s, 1H), 8.57 (s,2H), 8.29 (s, 1H), 7.52-7.55 (d, J = 10.4 Hz, 1H), 6.0- 6.03 (d, J =10.4 Hz, 1H), 3.51-3.64 (m, 8H). LCMS m/z 507.25 [M + H]⁺, t_(R) = 2.012min 13

¹H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.83 (s, 1H), 9.56 (s, 1H),8.54-8.56 (m, 2H), 8.25-8.30(m, 1H), 7.49-7.51 (d, J = 10.4 Hz, 1H) ),6.01-6.04 (d, J = 10.4 Hz, 1H), 3.44-3.57 (m, 2H), 3.28-3.34 (m, 2H),3.21 (s, 1H), 3.15 (s, 1H), 2.84-2.88 (m, 2H), 0.93-1.04(m, 6H): LCMSm/z 521.18 [M + H]⁺, t_(R) 1.898 min 14

¹H NMR (400 MHz, DMSO-d6) δ 10.33 (bs, 2H), 9.63 (s, 1H), 8.57 (s, 2H),8.30 (s, 1H), 7.50-7.52 (d, J = 8 Hz, 1H) ), 6.01- 6.03 (d, J = 8 Hz,1H), 4.08-4.12 (m, 4H), 3.85-3.87 (m, 2H), 3.41-3.44 (m, 2H). LCMS m/z507.13 [M + H]⁺, t_(R) 1.950 min 15

¹H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.81 (s, 1H), 9.62 (s, 1H),8.56 (s, 2H), 8.29 (s, 1H), 7.49-7.51 (d, J = 10.4 Hz, 1H) ), 6.01-6.03(d, J = 10.4 Hz, 1H), 3.65-3.67 (m, 2H), 3.30-3.34 (m, 2H), 3.08 (bs,2H), 2.55-2.58 (m, 2H), 0.96 (s, 6H). LCMS m/z 521.18 [M + H]⁺, t_(R)1.937 min

In some embodiments, the compound of the invention is selected from anyone of compounds 1 to 11. In one aspect of these embodiments, thecompound is selected from any one of compounds 1, 2, 3, 4, 8 and 11.

Pharmacokinetics (PK) play an increasing role in drug discovery anddevelopment. Pharmacokinetics is the quantitative study of the timecourse of drug absorption, distribution, metabolism and/or excretion.When a drug is administered, it distributes rapidly from itsadministration site into the systemic blood circulation. One measure ofthe extent of a therapeutic agent's distribution is the area under theplasma concentration-time curve (AUC), calculated to the last measuredconcentration (AUC_(t)) and extrapolated to infinity (AUC_(Inf)). AUC isthus a frequently used metric to quantitate drug exposure.

In general, the higher the exposure of a therapeutic agent, the greaterthe effects of the agent. However, high exposure of a therapeutic agentmay have deleterious effects on certain tissues such as the brain. Whilethe blood-brain barrier (BBB), a protective network consisting of tightjunctions between endothelial cells, restricts the diffusion ofhydrophilic and/or large molecules, drugs with high AUC are stillcapable of penetrating the BBB and/or cerebrospinal fluid. Suchpenetration is often undesirable and can lead to unwanted side effects.Current drug discovery efforts are aimed, in part, at striking a balancebetween maximizing drug exposure (i.e. AUC) while minimizing brainpenetration.

The brain to plasma (B:P) ratio is once such method of quantifying therelative distribution of a therapeutic agent in brain tissue to that incirculation. Such a ratio provides one indication of the brainpenetration of a given therapeutic agent. A high brain to plasma ratiois preferred when targeting diseases localized in the central nervoussystem (CNS), including the brain and the cerebrospinal fluid. However,a lower brain to plasma ratio is generally preferable for non-CNStherapeutic agents to minimize brain penetration to avoid potential sideeffects. Thus, a low brain to plasma ratio is preferable to avoidunwanted accumulation of therapeutic agents in the brain and CNS tissue.

As set forth in more detail in the Example section, the compounds of thepresent invention display a higher AUC and/or a lower B:P as compared toother nuclear transport inhibitors, such as those disclosed in co-ownedU.S. patent application Ser. No. 13/041,377, filed Mar. 5, 2011 andpublished as US 2009/0275607 on Nov. 10, 2011. In some embodiments, thepresent invention provides a compound of formula I, wherein the compoundhas <1 μM (less than 1 μM) nuclear export activity, an AUC_(Inf) ofgreater than about 3500; and a B:P of less than about 2.5 when dosed ina mouse at 10 mg/kg po.

The novel features of the present invention will become apparent tothose of skill in the art upon examination of the following detaileddescription of the invention. It should be understood, however, that thedetailed description of the invention and the specific examplespresented, while indicating certain embodiments of the presentinvention, are provided for illustration purposes only because variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those of skill in the art from the detaileddescription of the invention and claims that follow.

Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

Unless specified otherwise within this specification, the nomenclatureused in this specification generally follows the examples and rulesstated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F,and H, Pergamon Press, Oxford, 1979, which is incorporated by referenceherein for its exemplary chemical structure names and rules on namingchemical structures. Optionally, a name of a compound may be generatedusing a chemical naming program: ACD/ChemSketch, Version 5.09/September2001, Advanced Chemistry Development, Inc., Toronto, Canada.

Compounds of the present invention may have asymmetric centers, chiralaxes, and chiral planes (e.g., as described in: E. L. Eliel and S. H.Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, NewYork, 1994, pages 1119-1190), and occur as racemates, racemic mixtures,and as individual diastereomers or enantiomers, with all possibleisomers and mixtures thereof, including optical isomers, being includedin the present invention.

The term “halo” or “halogen” as used herein means halogen and includes,for example, and without being limited thereto, fluoro, chloro, bromo,iodo and the like, in both radioactive and non-radioactive forms.

The term “alkyl,” as used herein, unless otherwise indicated, meansstraight or branched saturated monovalent hydrocarbon radicals,typically C₁-C₁₂, preferably C₁-C₆. As such, “C₁-C₆ alkyl” means astraight or branched saturated monovalent hydrocarbon radical havingfrom one to six carbon atoms (e.g., 1, 2, 3, 4, 5 or 6). Examples ofalkyl groups include, but are not limited to, methyl, ethyl, propyl,isopropyl, and t-butyl.

It is understood that substituents and substitution patterns on thecompounds of the invention can be selected by one of ordinary skill inthe art to provide compounds that are chemically stable and that can bereadily synthesized by techniques known in the art, as well as thosemethods set forth below. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substitutedgroup” can have a suitable substituent at each substitutable position ofthe group and, when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent can be either the same or different at everyposition. Alternatively, an “optionally substituted group” can beunsubstitued.

Combinations of substituents envisioned by this invention are preferablythose that result in the formation of stable or chemically feasiblecompounds. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups can be on the samecarbon atom or on different carbon atoms, as long as a stable structureresults. The term “stable,” as used herein, refers to compounds that arenot substantially altered when subjected to conditions to allow fortheir production, detection, and, in certain embodiments, theirrecovery, purification, and use for one or more of the purposesdisclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted group” are independently halogen;—(CH₂)₀₋₄R^(o); —(CH₂)₀₋₄OR^(o); —O(CH₂)₀₋₄R, —O—(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄CH(OR^(o))₂; —(CH₂)₀₋₄SR^(o); —(CH₂)₀₋₄Ph, which may besubstituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(o); —CH═CHPh, which may be substituted with R^(o);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(o); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(o))₂; —(CH₂)₀₋₄N(R^(o))C(O)R^(o);—N(R^(o))C(S)R^(o); —(CH₂)₀₋₄N(R^(o))C(O)NR^(o) ₂; —N(R^(o))C(S)NR^(o)₂; —(CH₂)₀₋₄N(R^(o))C(O)OR^(o); —N(R^(o))N(R^(o))C(O)R^(o);—N(R^(o))N(R^(o))C(O)NR^(o) ₂; —N(R^(o))N(R^(o))C(O)OR^(o);—(CH₂)₀₋₄C(O)R^(o); —C(S)R^(o); —(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄C(O)SR^(o); —(CH₂)₀₋₄C(O)OSiR^(o) ₃; —(CH₂)₀₋₄OC(O)R^(o);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(o); —(CH₂)₀₋₄SC(O)R^(o); —(CH₂)₀₋₄C(O)NR^(o)₂; —C(S)NR^(o) ₂; —C(S)SR^(o); —SC(S)SR^(o), —(CH₂)₀₋₄OC(O)NR^(o) ₂;—C(O)N(OR^(o))R^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o);—C(NOR^(o))R^(o); —(CH₂)₀₋₄ SSR^(o); —(CH₂)₀₋₄S(O)₂R^(o);—(CH₂)₀₋₄S(O)₂OR^(o); —(CH₂)₀₋₄₀S(O)₂R^(o); —S(O)₂NR^(o) ₂;—(CH₂)₀₋₄S(O)R^(o); —N(R^(o))S(O)₂NR^(o) ₂; —N(R^(o))S(O)₂R^(o);—N(OR^(o))R^(o); —C(NH)NR^(o) ₂; —P(O)₂R^(o); —P(O)R^(o) ₂; —OP(O)R^(o)₂; —OP(O)(OR^(o))₂; SiR^(o) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(o))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(o))₂, wherein each R^(o) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(o), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl monocyclic orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, and sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(o) (or the ring formed by takingtwo independent occurrences of R^(o) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. Suitable divalent substituents on asaturated carbon atom of R^(o) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted group” include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, and—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, and —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted group” include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, and —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmonocyclic or bicyclic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur.

Preferred substituents on heteroaryl can be selected from the groupconsisting of —OH, —SH, nitro, halogen, amino, cyano, C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, C₁-C₁₂ haloalkyl, C₁-C₁₂haloalkoxy and C₁-C₁₂ alkyl sulfanyl. Preferred substituents on alkyl,alkylene and heterocyclyl include the preferred substituents onheteroaryl and oxo. In one embodiment, the substituent on an alkyl,alkylene, heterocyclyl or heteroaryl is an amino group having theformula —N(R⁵)₂, wherein each R⁵ is independently selected from hydrogenand C₁-C₄ alkyl.

Substituents on alkyl, aklylene, heterocyclyl and heteroaryl can beselected from —OH, —SH, nitro, halogen, amino, cyano, C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl group, C₁-C₁₂ alkoxy, C₁-C₁₂ haloalkyl,C₁-C₁₂ haloalkoxy and C₁-C₁₂ alkyl sulfanyl. In one embodiment, thesubstituent is an amino group having the formula —N(R⁵)₂, wherein eachR⁵ is independently selected from hydrogen and C₁-C₄ alkyl.

The term “cycloalkyl”, as used herein, means saturated cyclichydrocarbons, i.e. compounds where all ring atoms are carbons. Examplesof cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and cycloheptyl. In some embodiments,cycloalkyl can optionally be substituted with one or more moresubstituents selected from from —OH, —SH, halogen, amino, nitro, cyano,C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl or C₂-C₁₂ alkynyl group, C₁-C₁₂ alkoxy,C₁-C₁₂ haloalkyl, and C₁-C₁₂ haloalkoxy.

The term “heteroaryl”, as used herein, refers to aromatic groupscontaining one or more heteroatoms (O, S, or N). A heteroaryl group canbe monocyclic or polycyclic, e.g. a monocyclic heteroaryl ring fused toone or more carbocyclic aromatic groups or other monocyclic heteroarylgroups. The heteroaryl groups of this invention can also include ringsystems substituted with one or more oxo moieties. Examples ofheteroaryl groups include, but are not limited to, pyridinyl,pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl,quinolyl, isoquinolyl, tetrazolyl, furyl, thienyl, isoxazolyl,thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl,indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl,indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, purinyl,oxadiazolyl, thiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzotriazolyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, dihydroquinolyl,tetrahydroquinolyl, dihydroisoquinolyl, tetrahydroisoquinolyl,benzofuryl, furopyridinyl, pyrolopyrimidinyl, and azaindolyl.

The foregoing heteroaryl groups may be C-attached or N-attached (wheresuch is possible). For instance, a group derived from pyrrole may bepyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached).

“Heterocyclyl” means a cyclic 4-13 membered saturated or unsaturatedaliphatic ring containing 1, 2, 3, 4 or 5 heteroatoms independentlyselected from N, O or S. When one heteroatom is S, it can be optionallymono- or di-oxygenated (i.e. —S(O)— or —S(O)₂—). The heterocyclyl can bemonocyclic, fused bicyclic, bridged bicyclic, spiro bicyclic orpolycyclic.

“Oxo” means ═O.

As used herein, the term “alkenyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 2 to 12 carbon atoms andhaving at least one carbon-carbon double bond. Alkenyl groups may beoptionally substituted with one or more substituents.

As used herein, the term “alkynyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 2 to 12 carbon atoms andhaving at least one carbon-carbon triple bond. Alkynyl groups may beoptionally substituted with one or more substituents.

As used herein, the term “alkylene” refers to an alkyl group that hastwo points of attachment to the rest of the compound. Non-limitingexamples of alkylene groups include methylene (—CH2-), ethylene(—CH2CH2-), n-propylene (—CH2CH2CH2-), isopropylene (—CH2CH(CH3)-), andthe like. Alkylene groups may be optionally substituted with one or moresubstituents.

The term “haloalkyl”, as used herein, includes an alkyl substituted withone or more F, Cl, Br, or I, wherein alkyl is defined above.

The terms “alkoxy”, as used herein, means an “alkyl-O—” group, whereinalkyl is defined above. Examples of alkoxy group include methoxy orethoxy groups.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,trifluoroacetic acid (2,2,2-trifluoroacetic acid), oxalic acid, maleicacid, tartaric acid, citric acid, succinic acid or malonic acid or byusing other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, trifluoroacetate(2,2,2-trifluoroacetate), undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

The term “pharmaceutically acceptable salt” means either an acidaddition salt or a basic addition salt which is compatible with thetreatment of patients.

In some embodiments, exemplary inorganic acids which form suitable saltsinclude, but are not limited thereto, hydrochloric, hydrobromic,sulfuric and phosphoric acid and acid metal salts such as sodiummonohydrogen orthophosphate and potassium hydrogen sulfate. Illustrativeorganic acids which form suitable salts include the mono-, di- andtricarboxylic acids. Illustrative of such acids are, for example,acetic, trifluoroacetic acid (2,2,2-trifluoroacetic acid), glycolic,lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric,citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic,phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic, p-toluenesulfonicacid and other sulfonic acids such as methanesulfonic acid and2-hydroxyethanesulfonic acid. Either the mono- or di-acid salts can beformed, and such salts can exist in either a hydrated, solvated orsubstantially anhydrous form. In general, the acid addition salts ofthese compounds are more soluble in water and various hydrophilicorganic solvents, and generally demonstrate higher melting points incomparison to their free base forms. Other non-pharmaceuticallyacceptable salts e.g. oxalates may be used for example in the isolationof compounds of Formula I for laboratory use, or for subsequentconversion to a pharmaceutically acceptable acid addition salt.

A “pharmaceutically acceptable basic addition salt” is any non-toxicorganic or inorganic base addition salt of the acid compoundsrepresented by Formula I or any of its intermediates. Illustrativeinorganic bases which form suitable salts include, but are not limitedthereto, lithium, sodium, potassium, calcium, magnesium or bariumhydroxides. Illustrative organic bases which form suitable salts includealiphatic, alicyclic or aromatic organic amines such as methylamine,trimethyl amine and picoline or ammonia. The selection of theappropriate salt may be important so that an ester functionality, ifany, elsewhere in the molecule is not hydrolyzed. The selection criteriafor the appropriate salt will be known to one skilled in the art.

Acid addition salts of the compounds of Formula I are most suitablyformed from pharmaceutically acceptable acids, and include for examplethose formed with inorganic acids e.g. hydrochloric, sulphuric orphosphoric acids and organic acids e.g. succinic, maleic, acetic,trifluoroacetic or fumaric acid. Other non-pharmaceutically acceptablesalts e.g. oxalates may be used for example in the isolation ofcompounds of Formula I for laboratory use, or for subsequent conversionto a pharmaceutically acceptable acid addition salt. Also includedwithin the scope of the invention are base addition salts (such assodium, potassium and ammonium salts), solvates and hydrates ofcompounds of the invention. The conversion of a given compound salt to adesired compound salt is achieved by applying standard techniques, wellknown to one skilled in the art.

The term “stereoisomers” is a general term for all isomers of theindividual molecules that differ only in the orientation of their atomsin space. It includes mirror image isomers (enantiomers), geometric(cis/trans) isomers and isomers of compounds with more than one chiralcentre that are not mirror images of one another (diastereomers).

The term “treat” or “treating” means to alleviate symptoms, eliminatethe causation of the symptoms either on a temporary or permanent basis,or to prevent or slow the appearance of symptoms of the named disorderor condition.

The term “therapeutically effective amount” means an amount of thecompound which is effective in treating or lessening the severity of oneor more symptoms of a disorder or condition.

When introducing elements disclosed herein, the articles “a”, “an”,“the”, and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “having”, “including” are intended tobe open-ended and mean that there may be additional elements other thanthe listed elements.

Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit CRM1, in a biologicalsample or in a patient. In certain embodiments, a composition of thisinvention is formulated for administration to a patient in need of suchcomposition. The term “patient”, as used herein, means an animal. Insome embodiments, the animal is a mammal. In certain embodiments, thepatient is a veterinary patient (i.e., a non-human mammal patient). Insome embodiments, the patient is a dog. In other embodiments, thepatient is a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Compositions of the present invention may be administered orally,parenterally (including subcutaneous, intramuscular, intravenous andintradermal), by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. In some embodiments,provided compounds or compositions are administrable intravenouslyand/or intraperitoneally.

The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intraocular, intravitreal, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intraperitonealintralesional and intracranial injection or infusion techniques.Preferably, the compositions are administered orally, subcutaneously,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. In some embodiments, aprovided oral formulation is formulated for immediate release orsustained/delayed release. In some embodiments, the

composition is suitable for buccal or sublingual administration,including tablets, lozenges and pastilles. A provided compound can alsobe in micro-encapsulated form.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. Pharmaceutically acceptable compositions of thisinvention may also be administered topically, especially when the targetof treatment includes areas or organs readily accessible by topicalapplication, including diseases of the eye, the skin, or the lowerintestinal tract. Suitable topical formulations are readily prepared foreach of these areas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions or in an ointment such aspetrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation.

In some embodiments, pharmaceutically acceptable compositions of thisinvention are formulated for intra-peritoneal administration.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. In one embodiment, provided compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe inhibitor can be administered to a patient receiving thesecompositions. In another embodiment, the dosage is from about 0.5 toabout 100 mg/kg of body weight, or between 1 mg and 1000 mg/dose, every4 to 120 hours, or according to the requirements of the particular drug.Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to about 6 times per day.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of CRM1 and are therefore useful for treating one or moredisorders associated with activity of CRM1. Thus, in certainembodiments, the present invention provides a method for treating aCRM1-mediated disorder comprising the step of administering to a patientin need thereof a compound of the present invention, or pharmaceuticallyacceptable composition thereof. The compounds and compositions describedherein can also be administered to cells in culture, e.g. in vitro or exvivo, or to a subject, e.g., in vivo, to treat, prevent, and/or diagnosea variety of disorders, including those described herein below.

The activity of a compound utilized in this invention as an inhibitor ofCRM1 may be assayed in vitro, in vivo or in a cell line. Detailedconditions for assaying a compound utilized in this invention as aninhibitor of CRM1 are set forth in the Examples below.

As used herein, the term “CRM1-mediated” disorder or condition, as usedherein, means any disease or other deleterious condition in which CRM1is known to play a role. Accordingly, another embodiment of the presentinvention relates to treating or lessening the severity of one or morediseases in which CRM1 is known to play a role. In some embodiments, thepresent invention provides methods of treating a disease associated withexpression or activity of p53, p73, p21, pRB, p27, IκB, NFκB, c-Abl,FOXO proteins, COX-2, or an HDAC (histone deacetylases) in a subjectcomprising administering to the patient a therapeutically effectiveamount of a compound described herein. In another embodiment, thepresent invention relates to a method of treating or lessening theseverity of a disease or condition selected from a proliferativedisorder (e.g., cancer), an inflammatory disorder, an autoimmunedisorder, a viral infection, an ophthalmological disorder or aneurodegenerative disorder wherein said method comprises administeringto a patient in need thereof a compound or composition according to thepresent invention. In a more specific embodiment, the present inventionrelates to a method of treating or lessening the severity of cancer.Specific examples of the above disorders are set forth in detail below.

Cancers treatable by the compounds of this invention include, but arenot limited to, hematologic malignancies (leukemias, lymphomas, myelomasincluding multiple myeloma, myelodysplastic and myeloproliferativesyndromes) and solid tumors (carcinomas such as prostate, breast, lung,colon, pancreatic, renal, ovarian as well as soft tissue andosteosarcomas, and stromal tumors). Breast cancer (BC) can includebasal-like breast cancer (BLBC), triple negative breast cancer (TNBC)and breast cancer that is both BLBC and TNBC. In addition, breast cancercan include invasive or non-invasive ductal or lobular carcinoma,tubular, medullary, mucinous, papillary, cribriform carcinoma of thebreast, male breast cancer, recurrent or metastatic breast cancer,phyllodes tumor of the breast and Paget's disease of the nipple.

Inflammatory disorders treatable by the compounds of this inventioninclude, but are not limited to, multiple sclerosis, rheumatoidarthritis, degenerative joint disease, systemic lupus, systemicsclerosis, vasculitis syndromes (small, medium and large vessel),atherosclerosis, inflammatory bowel disease, irritable bowel syndrome,Crohn's disease, mucous colitis, ulcerative colitis, gastritis, sepsis,psoriasis and other dermatological inflammatory disorders (such aseczema, atopic dermatitis, contact dermatitis, urticaria, scleroderma,and dermatosis with acute inflammatory components, pemphigus,pemphigoid, allergic dermatitis), and urticarial syndromes.

Viral diseases treatable by the compounds of this invention include, butare not limited to, acute febrile pharyngitis, pharyngoconjunctivalfever, epidemic keratoconjunctivitis, infantile gastroenteritis,Coxsackie infections, infectious mononucleosis, Burkitt lymphoma, acutehepatitis, chronic hepatitis, hepatic cirrhosis, hepatocellularcarcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children,tonsillitis and pharyngitis in adults, keratoconjunctivitis), latentHSV-1 infection (e.g., herpes labialis and cold sores), primary HSV-2infection, latent HSV-2 infection, aseptic meningitis, infectiousmononucleosis, Cytomegalic inclusion disease, Kaposi's sarcoma,multicentric Castleman disease, primary effusion lymphoma, AIDS,influenza, Reye syndrome, measles, postinfectious encephalomyelitis,Mumps, hyperplastic epithelial lesions (e.g., common, flat, plantar andanogenital warts, laryngeal papillomas, epidermodysplasiaverruciformis), cervical carcinoma, squamous cell carcinomas, croup,pneumonia, bronchiolitis, common cold, Poliomyelitis, Rabies,influenza-like syndrome, severe bronchiolitis with pneumonia, Germanmeasles, congenital rubella, Varicella, and herpes zoster. Viraldiseases treatable by the compounds of this invention also includechronic viral infections, including hepatitis B and hepatitis C.

Exemplary ophthalmology disorders include, but are not limited to,macular edema (diabetic and nondiabetic macular edema), aged relatedmacular degeneration wet and dry forms, aged disciform maculardegeneration, cystoid macular edema, palpebral edema, retina edema,diabetic retinopathy, chorioretinopathy, neovascular maculopathy,neovascular glaucoma, uveitis, iritis, retinal vasculitis,endophthalmitis, panophthalmitis, metastatic ophthalmia, choroiditis,retinal pigment epitheliitis, conjunctivitis, cyclitis, scleritis,episcleritis, optic neuritis, retrobulbar optic neuritis, keratitis,blepharitis, exudative retinal detachment, corneal ulcer, conjunctivalulcer, chronic nummular keratitis, ophthalmic disease associated withhypoxia or ischemia, retinopathy of prematurity, proliferative diabeticretinopathy, polypoidal choroidal vasculopathy, retinal angiomatousproliferation, retinal artery occlusion, retinal vein occlusion, Coats'disease, familial exudative vitreoretinopathy, pulseless disease(Takayasu's disease), Eales disease, antiphospholipid antibody syndrome,leukemic retinopathy, blood hyperviscosity syndrome, macroglobulinemia,interferon-associated retinopathy, hypertensive retinopathy, radiationretinopathy, corneal epithelial stem cell deficiency or cataract.

Neurodegenerative diseases treatable by a compound of Formula I include,but are not limited to, Parkinson's, Alzheimer's, and Huntington's, andAmyotrophic lateral sclerosis (ALS/Lou Gehrig's Disease).

Compounds and compositions described herein may also be used to treatdisorders of abnormal tissue growth and fibrosis including dilativecardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,pulmonary fibrosis, hepatic fibrosis, glomerulonephritis, polycystickidney disorder (PKD) and other renal disorders.

Compounds and compositions described herein may also be used to treatdisorders related to food intake such as obesity and hyperphagia.

In another embodiment, a compound or composition described herein may beused to treat or prevent allergies and respiratory disorders, includingasthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygentoxicity, emphysema, chronic bronchitis, acute respiratory distresssyndrome, and any chronic obstructive pulmonary disease (COPD).

In some embodiments, the disorder or condition associated with CRM1activity is muscular dystrophy, arthritis, for example, osteoarthritisand rheumatoid arthritis, ankylosing spondilitis, traumatic braininjury, spinal cord injury, sepsis, rheumatic disease, canceratherosclerosis, type 1 diabetes, type 2 diabetes, leptospiriosis renaldisease, glaucoma, retinal disease, ageing, headache, pain, complexregional pain syndrome, cardiac hypertrophy, musclewasting, catabolicdisorders, obesity, fetal growth retardation, hypercholesterolemia,heart disease, chronic heart failure, ischemia/reperfusion, stroke,cerebral aneurysm, angina pectoris, pulmonary disease, cystic fibrosis,acid-induced lung injury, pulmonary hypertension, asthma, chronicobstructive pulmonary disease, Sjogren's syndrome, hyaline membranedisease, kidney disease, glomerular disease, alcoholic liver disease,gut diseases, peritoneal endometriosis, skin diseases, nasal sinusitis,mesothelioma, anhidrotic ecodermal dysplasia-ID, behcet's disease,incontinentia pigmenti, tuberculosis, asthma, crohn's disease, colitis,ocular allergy, appendicitis, paget's disease, pancreatitis,periodonitis, endometriosis, inflammatory bowel disease, inflammatorylung disease, silica-induced diseases, sleep apnea, AIDS, HIV-1,autoimmune diseases, antiphospholipid syndrome, lupus, lupus nephritis,familial mediterranean fever, hereditary periodic fever syndrome,psychosocial stress diseases, neuropathological diseases, familialamyloidotic polyneuropathy, inflammatory neuropathy, parkinson'sdisease, multiple sclerosis, alzheimer's disease, amyotropic lateralsclerosis, huntington's disease, cataracts, or hearing loss.

In other embodiments, the disorder or condition associated with CRM1activity is head injury, uveitis, inflammatory pain, allergen inducedasthma, non-allergen induced asthma, glomerular nephritis, ulcerativecolitis, necrotizing enterocolitis, hyperimmunoglobulinemia D withrecurrent fever (HIDS), TNF receptor associated periodic syndrome(TRAPS), cryopyrin-associated periodic syndromes, Muckle-Wells syndrome(urticaria deafness amyloidosis), familial cold urticaria, neonatalonset multisystem inflammatory disease (NOMID), periodic fever, aphthousstomatitis, pharyngitis and adenitis (PFAPA syndrome), Blau syndrome,pyogenic sterile arthritis, pyoderma gangrenosum, acne (PAPA),deficiency of the interleukin-1-receptor antagonist (DIRA), subarachnoidhemorrhage, polycystic kidney disease, transplant, organ transplant,tissue transplant, myelodysplastic syndrome, irritant-inducedinflammation, plant irritant-induced inflammation, poison ivy/urushioloil-induced inflammation, chemical irritant-induced inflammation, beesting-induced inflammation, insect bite-induced inflammation, sunburn,burns, dermatitis, endotoxemia, lung injury, acute respiratory distresssyndrome, alcoholic hepatitis, or kidney injury caused by parasiticinfections.

In further aspects, the present invention provides a use of a compoundof formula I for the manufacture of a medicament for the treatment of adisease associated with expression or activity of p53, p73, p21, pRB,p27, IκB, NFκB, c-Abl, FOXO proteins, COX-2 or an HDAC in a subject. Insome embodiments, the present invention provides a use of a compound offormula I in the manufacture of a medicament for the treatment of any ofcancer and/or neoplastic disorders, angiogenesis, autoimmune disorders,inflammatory disorders and/or diseases, epigenetics, hormonal disordersand/or diseases, viral diseases, neurodegenerative disorders and/ordiseases, wounds, and ophthalmologic disorders.

In some embodiments, the present invention provides a method forinhibiting CRM1 in a biological sample comprising contacting thebiological sample with, or administering to the patient, apharmaceutically acceptable salt of a compound of Formula I, orpharmaceutically acceptable composition thereof.

Neoplastic Disorders

A compound or composition described herein can be used to treat aneoplastic disorder. A “neoplastic disorder” is a disease or disordercharacterized by cells that have the capacity for autonomous growth orreplication, e.g., an abnormal state or condition characterized byproliferative cell growth. Exemplary neoplastic disorders include:carcinoma, sarcoma, metastatic disorders, e.g., tumors arising fromprostate, brain, bone, colon, lung, breast, ovarian, and liver origin,hematopoietic neoplastic disorders, e.g., leukemias, lymphomas, myelomaand other malignant plasma cell disorders, and metastatic tumors.Prevalent cancers include: breast, prostate, colon, lung, liver, andpancreatic cancers. Treatment with the compound can be in an amounteffective to ameliorate at least one symptom of the neoplastic disorder,e.g., reduced cell proliferation, reduced tumor mass, etc.

The disclosed methods are useful in the prevention and treatment ofcancer, including for example, solid tumors, soft tissue tumors, andmetastases thereof, as well as in familial cancer syndromes such as LiFraumeni Syndrome, Familial Breast-Ovarian Cancer (BRCA1 or BRAC2mutations) Syndromes, and others. The disclosed methods are also usefulin treating non-solid cancers. Exemplary solid tumors includemalignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of thevarious organ systems, such as those of lung, breast, lymphoid,gastrointestinal (e.g., colon), and genitourinary (e.g., renal,urothelial, or testicular tumors) tracts, pharynx, prostate, and ovary.Exemplary adenocarcinomas include colorectal cancers, renal-cellcarcinoma, liver cancer, non-small cell carcinoma of the lung, andcancer of the small intestine.

Exemplary cancers described by the National Cancer Institute include:Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia,Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma;Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS-RelatedMalignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar;Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; BladderCancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/MalignantFibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult;Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, CerebellarAstrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/MalignantGlioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor,Medulloblastoma, Childhood; Brain Tumor, Supratentorial PrimitiveNeuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway andHypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); BreastCancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; BreastCancer, Male; Bronchial Adenomas/Carcinoids, Childhood; Carcinoid Tumor,Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical;Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central NervousSystem Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; CerebralAstrocytoma/Malignant Glioma, Childhood; Cervical Cancer; ChildhoodCancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia;Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of TendonSheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-CellLymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer,Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Familyof Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal GermCell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, IntraocularMelanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric(Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; GastrointestinalCarcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ CellTumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational TrophoblasticTumor; Glioma, Childhood Brain Stem; Glioma, Childhood Visual Pathwayand Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer;Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver)Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin'sLymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; HypopharyngealCancer; Hypothalamic and Visual Pathway Glioma, Childhood; IntraocularMelanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma;Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia,Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood;Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood;Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia,Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary);Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; LungCancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; LymphoblasticLeukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma,AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma,Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's,Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma,Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma,Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central NervousSystem; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; MalignantMesothelioma, Adult; Malignant Mesothelioma, Childhood; MalignantThymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular;Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous NeckCancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome,Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides;Myelodysplastic Syndromes; Myelogenous Leukemia, Chronic; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple; MyeloproliferativeDisorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma;Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood;Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer;Oral Cancer, Childhood; Oral Cavity and Lip Cancer; OropharyngealCancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; OvarianCancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor;Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; PancreaticCancer, Childhood; Pancreatic Cancer, Islet Cell; Paranasal Sinus andNasal Cavity Cancer; Parathyroid Cancer; Penile Cancer;Pheochromocytoma; Pineal and Supratentorial Primitive NeuroectodermalTumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer;Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma;Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult;Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; RenalCell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis andUreter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma,Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood;Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma(Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma,Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, SoftTissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood;Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell LungCancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft TissueSarcoma, Childhood; Squamous Neck Cancer with Occult Primary,Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer,Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood;T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood;Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood;Transitional Cell Cancer of the Renal Pelvis and Ureter; TrophoblasticTumor, Gestational; Unknown Primary Site, Cancer of, Childhood; UnusualCancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer;Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway andHypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor. Metastases of the aforementioned cancerscan also be treated or prevented in accordance with the methodsdescribed herein.

Combination Therapies

In some embodiments, a compound described herein is administeredtogether with an additional “second” therapeutic agent or treatment. Thechoice of second therapeutic agent may be made from any agent that istypically used in a monotherapy to treat the indicated disease orcondition. As used herein, the term “administered together” and relatedterms refers to the simultaneous or sequential administration oftherapeutic agents in accordance with this invention. For example, acompound of the present invention may be administered with anothertherapeutic agent simultaneously or sequentially in separate unit dosageforms or together in a single unit dosage form. Accordingly, the presentinvention provides a single unit dosage form comprising a compound offormula I, an additional therapeutic agent, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.

In one embodiment of the invention, where a second therapeutic agent isadministered to a subject, the effective amount of the compound of thisinvention is less than its effective amount would be where the secondtherapeutic agent is not administered. In another embodiment, theeffective amount of the second therapeutic agent is less than itseffective amount would be where the compound of this invention is notadministered. In this way, undesired side effects associated with highdoses of either agent may be minimized. Other potential advantages(including without limitation improved dosing regimens and/or reduceddrug cost) will be apparent to those of skill in the art. The additionalagents may be administered separately, as part of a multiple doseregimen, from the compounds of this invention. Alternatively, thoseagents may be part of a single dosage form, mixed together with thecompounds of this invention in a single composition.

Cancer Combination Therapies

In some embodiments, a compound described herein is administeredtogether with an additional cancer treatment. Exemplary additionalcancer treatments include, for example: chemotherapy, targeted therapiessuch as antibody therapies, kinase inhibitors, immunotherapy, andhormonal therapy, epigenetic therapy, proteosome inhibitors, andanti-angiogenic therapies. Examples of each of these treatments areprovided below. As used herein, the term “combination,” “combined,” andrelated terms refer to the simultaneous or sequential administration oftherapeutic agents in accordance with this invention. For example, acompound of the present invention can be administered with anothertherapeutic agent simultaneously or sequentially in separate unit dosageforms or together in a single unit dosage form. Accordingly, the presentinvention provides a single unit dosage form comprising a compound ofthe invention, an additional therapeutic agent, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.

The amount of both a compound of the invention and additionaltherapeutic agent (in those compositions which comprise an additionaltherapeutic agent as described above) that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration.Preferably, compositions of this invention should be formulated so thata dosage of between 0.01-100 mg/kg body weight/day of a compound of theinvention can be administered.

Chemotherapy

In some embodiments, a compound described herein is administered with achemotherapy. Chemotherapy is the treatment of cancer with drugs thatcan destroy cancer cells. “Chemotherapy” usually refers to cytotoxicdrugs which affect rapidly dividing cells in general, in contrast withtargeted therapy. Chemotherapy drugs interfere with cell division invarious possible ways, e.g., with the duplication of DNA or theseparation of newly formed chromosomes. Most forms of chemotherapytarget all rapidly dividing cells and are not specific for cancer cells,although some degree of specificity may come from the inability of manycancer cells to repair DNA damage, while normal cells generally can.

Examples of chemotherapeutic agents used in cancer therapy include, forexample, antimetabolites (e.g., folic acid, purine, and pyrimidinederivatives) and alkylating agents (e.g., nitrogen mustards,nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes,aziridines, spindle poison, cytotoxic agents, topoisomerase inhibitorsand others). Exemplary agents include Aclarubicin, Actinomycin,Alitretinoin, Altretamine, Aminopterin, Aminolevulinic acid, Amrubicin,Amsacrine, Anagrelide, Arsenic trioxide, Asparaginase, Atrasentan,Belotecan, Bexarotene, Bendamustin, Bleomycin, Bortezomib, Busulfan,Camptothecin, Capecitabine, Carboplatin, Carboquone, Carmofur,Carmustine, Celecoxib, Chlorambucil, Chlormethine, Cisplatin,Cladribine, Clofarabine, Crisantaspase, Cyclophosphamide, Cytarabine,Dacarbazine, Dactinomycin, Daunorubicin, Decitabine, Demecolcine,Docetaxel, Doxorubicin, Efaproxiral, Elesclomol, Elsamitrucin,Enocitabine, Epirubicin, Estramustine, Etoglucid, Etoposide,Floxuridine, Fludarabine, Fluorouracil (5FU), Fotemustine, Gemcitabine,Gliadel implants, Hydroxycarbamide, Hydroxyurea, Idarubicin, Ifosfamide,Irinotecan, Irofulven, Ixabepilone, Larotaxel, Leucovorin, Liposomaldoxorubicin, Liposomal daunorubicin, Lonidamine, Lomustine, Lucanthone,Mannosulfan, Masoprocol, Melphalan, Mercaptopurine, Mesna, Methotrexate,Methyl aminolevulinate, Mitobronitol, Mitoguazone, Mitotane, Mitomycin,Mitoxantrone, Nedaplatin, Nimustine, Oblimersen, Omacetaxine, Ortataxel,Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed, Pentostatin,Pirarubicin, Pixantrone, Plicamycin, Porfimer sodium, Prednimustine,Procarbazine, Raltitrexed, Ranimustine, Rubitecan, Sapacitabine,Semustine, Sitimagene ceradenovec, Strataplatin, Streptozocin,Talaporfin, Tegafur-uracil, Temoporfin, Temozolomide, Teniposide,Tesetaxel, Testolactone, Tetranitrate, Thiotepa, Tiazofurine,Tioguanine, Tipifarnib, Topotecan, Trabectedin, Triaziquone,Triethylenemelamine, Triplatin, Tretinoin, Treosulfan, Trofosfamide,Uramustine, Valrubicin, Verteporfin, Vinblastine, Vincristine,Vindesine, Vinflunine, Vinorelbine, Vorinostat, Zorubicin, and othercytostatic or cytotoxic agents described herein.

Because some drugs work better together than alone, two or more drugsare often given at the same time. Often, two or more chemotherapy agentsare used as combination chemotherapy. In some embodiments, thechemotherapy agents (including combination chemotherapy) can be used incombination with a compound described herein.

Targeted Therapy

Targeted therapy constitutes the use of agents specific for thederegulated proteins of cancer cells. Small molecule targeted therapydrugs are generally inhibitors of enzymatic domains on mutated,overexpressed, or otherwise critical proteins within the cancer cell.Prominent examples are the tyrosine kinase inhibitors such as Axitinib,Bosutinib, Cediranib, desatinib, erolotinib, imatinib, gefitinib,lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sorafenib, Sunitinib, andVandetanib, and also cyclin-dependent kinase inhibitors such asAlvocidib and Seliciclib. Monoclonal antibody therapy is anotherstrategy in which the therapeutic agent is an antibody whichspecifically binds to a protein on the surface of the cancer cells.Examples include the anti-HER2/neu antibody trastuzumab (Herceptin®)typically used in breast cancer, and the anti-CD20 antibody rituximaband Tositumomab typically used in a variety of B-cell malignancies.Other exemplary antibodies include Cetuximab, Panitumumab, Trastuzumab,Alemtuzumab, Bevacizumab, Edrecolomab, and Gemtuzumab. Exemplary fusionproteins include Aflibercept and Denileukin diftitox. In someembodiments, the targeted therapy can be used in combination with acompound described herein, e.g., Gleevec (Vignari and Wang 2001).

Targeted therapy can also involve small peptides as “homing devices”which can bind to cell surface receptors or affected extracellularmatrix surrounding the tumor. Radionuclides which are attached to thesepeptides (e.g., RGDs) eventually kill the cancer cell if the nuclidedecays in the vicinity of the cell. An example of such therapy includesBEXXAR®.

Angiogenesis

Compounds and methods described herein may be used to treat or prevent adisease or disorder associated with angiogenesis. Diseases associatedwith angiogenesis include cancer, cardiovascular disease and maculardegeneration.

Angiogenesis is the physiological process involving the growth of newblood vessels from pre-existing vessels. Angiogenesis is a normal andvital process in growth and development, as well as in wound healing andin granulation tissue. However, it is also a fundamental step in thetransition of tumors from a dormant state to a malignant one.Angiogenesis may be a target for combating diseases characterized byeither poor vascularisation or abnormal vasculature.

Application of specific compounds that may inhibit or induce thecreation of new blood vessels in the body may help combat such diseases.The presence of blood vessels where there should be none may affect themechanical properties of a tissue, increasing the likelihood of failure.The absence of blood vessels in a repairing or otherwise metabolicallyactive tissue may inhibit repair or other essential functions. Severaldiseases, such as ischemic chronic wounds, are the result of failure orinsufficient blood vessel formation and may be treated by a localexpansion of blood vessels, thus bringing new nutrients to the site,facilitating repair. Other diseases, such as age-related maculardegeneration, may be created by a local expansion of blood vessels,interfering with normal physiological processes.

Vascular endothelial growth factor (VEGF) has been demonstrated to be amajor contributor to angiogenesis, increasing the number of capillariesin a given network. Upregulation of VEGF is a major component of thephysiological response to exercise and its role in angiogenesis issuspected to be a possible treatment in vascular injuries. In vitrostudies clearly demonstrate that VEGF is a potent stimulator ofangiogenesis because, in the presence of this growth factor, platedendothelial cells will proliferate and migrate, eventually forming tubestructures resembling capillaries.

Tumors induce blood vessel growth (angiogenesis) by secreting variousgrowth factors (e.g., VEGF). Growth factors such as bFGF and VEGF caninduce capillary growth into the tumor, which some researchers suspectsupply required nutrients, allowing for tumor expansion.

Angiogenesis represents an excellent therapeutic target for thetreatment of cardiovascular disease. It is a potent, physiologicalprocess that underlies the natural manner in which our bodies respond toa diminution of blood supply to vital organs, namely the production ofnew collateral vessels to overcome the ischemic insult.

Overexpression of VEGF causes increased permeability in blood vessels inaddition to stimulating angiogenesis. In wet macular degeneration, VEGFcauses proliferation of capillaries into the retina. Since the increasein angiogenesis also causes edema, blood and other retinal fluids leakinto the retina, causing loss of vision.

Anti-angiogenic therapy can include kinase inhibitors targeting vascularendothelial growth factor (VEGF) such as sunitinib, sorafenib, ormonoclonal antibodies or receptor “decoys” to VEGF or VEGF receptorincluding bevacizumab or VEGF-Trap, or thalidomide or its analogs(lenalidomide, pomalidomide), or agents targeting non-VEGF angiogenictargets such as fibroblast growth factor (FGF), angiopoietins, orangiostatin or endostatin.

Epigenetics

Compounds and methods described herein may be used to treat or prevent adisease or disorder associated with epigenetics. Epigenetics is thestudy of heritable changes in phenotype or gene expression caused bymechanisms other than changes in the underlying DNA sequence. Oneexample of epigenetic changes in eukaryotic biology is the process ofcellular differentiation. During morphogenesis, stem cells become thevarious cell lines of the embryo which in turn become fullydifferentiated cells. In other words, a single fertilized egg cellchanges into the many cell types including neurons, muscle cells,epithelium, blood vessels etc. as it continues to divide. It does so byactivating some genes while inhibiting others.

Epigenetic changes are preserved when cells divide. Most epigeneticchanges only occur within the course of one individual organism'slifetime, but, if a mutation in the DNA has been caused in sperm or eggcell that results in fertilization, then some epigenetic changes areinherited from one generation to the next. Specific epigenetic processesinclude paramutation, bookmarking, imprinting, gene silencing, Xchromosome inactivation, position effect, reprogramming, transvection,maternal effects, the progress of carcinogenesis, many effects ofteratogens, regulation of histone modifications and heterochromatin, andtechnical limitations affecting parthenogenesis and cloning.

Exemplary diseases associated with epigenetics include ATR-syndrome,fragile X-syndrome, ICF syndrome, Angelman's syndrome, Prader-Willssyndrome, BWS, Rett syndrome, α-thalassaemia, cancer, leukemia,Rubinstein-Taybi syndrome and Coffin-Lowry syndrome.

The first human disease to be linked to epigenetics was cancer.Researchers found that diseased tissue from patients with colorectalcancer had less DNA methylation than normal tissue from the samepatients. Because methylated genes are typically turned off, loss of DNAmethylation can cause abnormally high gene activation by altering thearrangement of chromatin. On the other hand, too much methylation canundo the work of protective tumor suppressor genes.

DNA methylation occurs at CpG sites, and a majority of CpG cytosines aremethylated in mammals. However, there are stretches of DNA near promoterregions that have higher concentrations of CpG sites (known as CpGislands) that are free of methylation in normal cells. These CpG islandsbecome excessively methylated in cancer cells, thereby causing genesthat should not be silenced to turn off. This abnormality is thetrademark epigenetic change that occurs in tumors and happens early inthe development of cancer. Hypermethylation of CpG islands can causetumors by shutting off tumor-suppressor genes. In fact, these types ofchanges may be more common in human cancer than DNA sequence mutations.

Furthermore, although epigenetic changes do not alter the sequence ofDNA, they can cause mutations. About half of the genes that causefamilial or inherited forms of cancer are turned off by methylation.Most of these genes normally suppress tumor formation and help repairDNA, including 06-methylguanine-DNA methyltransferase (MGMT), MLH1cyclin-dependent kinase inhibitor 2B (CDKN2B), and RASSF1A. For example,hypermethylation of the promoter of MGMT causes the number of G-to-Amutations to increase.

Hypermethylation can also lead to instability of microsatellites, whichare repeated sequences of DNA. Microsatellites are common in normalindividuals, and they usually consist of repeats of the dinucleotide CA.Too much methylation of the promoter of the DNA repair gene MLH1 canmake a microsatellite unstable and lengthen or shorten it.Microsatellite instability has been linked to many cancers, includingcolorectal, endometrial, ovarian, and gastric cancers.

Fragile X syndrome is the most frequently inherited mental disability,particularly in males. Both sexes can be affected by this condition, butbecause males only have one X chromosome, one fragile X will impact themmore severely. Indeed, fragile X syndrome occurs in approximately 1 in4,000 males and 1 in 8,000 females. People with this syndrome havesevere intellectual disabilities, delayed verbal development, and“autistic-like” behavior.

Fragile X syndrome gets its name from the way the part of the Xchromosome that contains the gene abnormality looks under a microscope;it usually appears as if it is hanging by a thread and easily breakable.The syndrome is caused by an abnormality in the FMR1 (fragile X mentalretardation 1) gene. People who do not have fragile X syndrome have 6 to50 repeats of the trinucleotide CGG in their FMR1 gene. However,individuals with over 200 repeats have a full mutation, and they usuallyshow symptoms of the syndrome. Too many CGGs cause the CpG islands atthe promoter region of the FMR1 gene to become methylated; normally,they are not. This methylation turns the gene off, stopping the FMR1gene from producing an important protein called fragile X mentalretardation protein. Loss of this specific protein causes fragile Xsyndrome. Although a lot of attention has been given to the CGGexpansion mutation as the cause of fragile X, the epigenetic changeassociated with FMR1 methylation is the real syndrome culprit.

Fragile X syndrome is not the only disorder associated with mentalretardation that involves epigenetic changes. Other such conditionsinclude Rubenstein-Taybi, Coffin-Lowry, Prader-Willi, Angelman,Beckwith-Wiedemann, ATR-X, and Rett syndromes.

Epigenetic therapies include inhibitors of enzymes controllingepigenetic modifications, specifically DNA methyltransferases andhistone deacetylases, which have shown promising anti-tumorigeniceffects for some malignancies, as well as antisense oligonucleotides andsiRNA.

Immunotherapy

In some embodiments, a compound described herein is administered with animmunotherapy. Cancer immunotherapy refers to a diverse set oftherapeutic strategies designed to induce the patient's own immunesystem to fight the tumor. Contemporary methods for generating an immuneresponse against tumors include intravesicular BCG immunotherapy forsuperficial bladder cancer, prostate cancer vaccine Provenge, and use ofinterferons and other cytokines to induce an immune response in renalcell carcinoma and melanoma patients.

Allogeneic hematopoietic stem cell transplantation can be considered aform of immunotherapy, since the donor's immune cells will often attackthe tumor in a graft-versus-tumor effect. In some embodiments, theimmunotherapy agents can be used in combination with a compounddescribed herein.

Hormonal Therapy

In some embodiments, a compound described herein is administered with ahormonal therapy. The growth of some cancers can be inhibited byproviding or blocking certain hormones. Common examples ofhormone-sensitive tumors include certain types of breast and prostatecancers, as well as certain types of leukemia which respond to certainretinoids/retinoic acids. Removing or blocking estrogen or testosteroneis often an important additional treatment. In certain cancers,administration of hormone agonists, such as progestogens may betherapeutically beneficial. In some embodiments, the hormonal therapyagents can be used in combination with a compound described herein.

Hormonal therapy agents include the administration of hormone agonistsor hormone antagonists and include retinoids/retinoic acid, compoundsthat inhibit estrogen or testosterone, as well as administration ofprogestogens.

Inflammation and Autoimmune Disease

The compounds and methods described herein may be used to treat orprevent a disease or disorder associated with inflammation, particularlyin humans and other mammals. A compound described herein may beadministered prior to the onset of, at, or after the initiation ofinflammation. When used prophylactically, the compounds are preferablyprovided in advance of any inflammatory response or symptom.Administration of the compounds can prevent or attenuate inflammatoryresponses or symptoms. Exemplary inflammatory conditions include, forexample, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis,degenerative joint disease, spondouloarthropathies, other seronegativeinflammatory arthridities, polymyalgia rheumatica, various vasculidities(e.g., giant cell arteritis, ANCA+ vasculitis), gouty arthritis,systemic lupus erythematosus, juvenile arthritis, juvenile rheumatoidarthritis, osteoarthritis, osteoporosis, diabetes (e.g., insulindependent diabetes mellitus or juvenile onset diabetes), menstrualcramps, cystic fibrosis, inflammatory bowel disease, irritable bowelsyndrome, Crohn's disease, mucous colitis, ulcerative colitis,gastritis, esophagitis, pancreatitis, peritonitis, Alzheimer's disease,shock, ankylosing spondylitis, gastritis, conjunctivitis, pancreatis(acute or chronic), multiple organ injury syndrome (e.g., secondary tosepticemia or trauma), myocardial infarction, atherosclerosis, stroke,reperfusion injury (e.g., due to cardiopulmonary bypass or kidneydialysis), acute glomerulonephritis, thermal injury (i.e., sunburn),necrotizing enterocolitis, granulocyte transfusion associated syndrome,and/or Sjogren's syndrome. Exemplary inflammatory conditions of the skininclude, for example, eczema, atopic dermatitis, contact dermatitis,urticaria, schleroderma, psoriasis, and dermatosis with acuteinflammatory components.

In another embodiment, a compound or method described herein may be usedto treat or prevent allergies and respiratory conditions, includingasthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygentoxicity, emphysema, chronic bronchitis, acute respiratory distresssyndrome, and any chronic obstructive pulmonary disease (COPD). Thecompounds may be used to treat chronic hepatitis infection, includinghepatitis B and hepatitis C.

Additionally, a compound or method described herein may be used to treatautoimmune diseases and/or inflammation associated with autoimmunediseases, such as organ-tissue autoimmune diseases (e.g., Raynaud'ssyndrome), scleroderma, myasthenia gravis, transplant rejection,endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiplesclerosis, autoimmune thyroiditis, uveitis, systemic lupuserythematosis, Addison's disease, autoimmune polyglandular disease (alsoknown as autoimmune polyglandular syndrome), and Grave's disease.

In a particular embodiment, the compounds described herein can be usedto treat multiple sclerosis. In a specific aspect, the compound used totreat multiple sclerosis is Compound 1:(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazetidin-1-yl)prop-2-en-1-one).

Combination Therapy

In certain embodiments, a compound described herein may be administeredalone or in combination with other compounds useful for treating orpreventing inflammation. Exemplary anti-inflammatory agents include, forexample, steroids (e.g., Cortisol, cortisone, fludrocortisone,prednisone, 6[alpha]-methylprednisone, triamcinolone, betamethasone ordexamethasone), nonsteroidal antiinflammatory drugs (NSAIDS (e.g.,aspirin, acetaminophen, tolmetin, ibuprofen, mefenamic acid, piroxicam,nabumetone, rofecoxib, celecoxib, etodolac or nimesulide). In anotherembodiment, the other therapeutic agent is an antibiotic (e.g.,vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime,ceftriaxone, cefixime, rifampinmetronidazole, doxycycline orstreptomycin). In another embodiment, the other therapeutic agent is aPDE4 inhibitor (e.g., roflumilast or rolipram). In another embodiment,the other therapeutic agent is an antihistamine (e.g., cyclizine,hydroxyzine, promethazine or diphenhydramine). In another embodiment,the other therapeutic agent is an anti-malarial (e.g., artemisinin,artemether, artsunate, chloroquine phosphate, mefloquine hydrochloride,doxycycline hyclate, proguanil hydrochloride, atovaquone orhalofantrine). In one embodiment, the other compound is drotrecoginalfa.

Further examples of anti-inflammatory agents include, for example,aceclofenac, acemetacin, e-acetamidocaproic acid, acetaminophen,acetaminosalol, acetanilide, acetylsalicylic acid, S-adenosylmethionine,alclofenac, alclometasone, alfentanil, algestone, allylprodine,alminoprofen, aloxiprin, alphaprodine, aluminum bis(acetylsalicylate),amcinonide, amfenac, aminochlorthenoxazin, 3-amino-4-hydroxybutyricacid, 2-amino-4-picoline, aminopropylon, aminopyrine, amixetrine,ammonium salicylate, ampiroxicam, amtolmetin guacil, anileridine,antipyrine, antrafenine, apazone, beclomethasone, bendazac, benorylate,benoxaprofen, benzpiperylon, benzydamine, benzylmorphine, bermoprofen,betamethasone, betamethasone-17-valerate, bezitramide,[alpha]-bisabolol, bromfenac, p-bromoacetanilide, 5-bromosalicylic acidacetate, bromosaligenin, bucetin, bucloxic acid, bucolome, budesonide,bufexamac, bumadizon, buprenorphine, butacetin, butibufen, butorphanol,carbamazepine, carbiphene, caiprofen, carsalam, chlorobutanol,chloroprednisone, chlorthenoxazin, choline salicylate, cinchophen,cinmetacin, ciramadol, clidanac, clobetasol, clocortolone, clometacin,clonitazene, clonixin, clopirac, cloprednol, clove, codeine, codeinemethyl bromide, codeine phosphate, codeine sulfate, cortisone,cortivazol, cropropamide, crotethamide, cyclazocine, deflazacort,dehydrotestosterone, desomorphine, desonide, desoximetasone,dexamethasone, dexamethasone-21-isonicotinate, dexoxadrol,dextromoramide, dextropropoxyphene, deoxycorticosterone, dezocine,diampromide, diamorphone, diclofenac, difenamizole, difenpiramide,diflorasone, diflucortolone, diflunisal, difluprednate, dihydrocodeine,dihydrocodeinone enol acetate, dihydromorphine, dihydroxyaluminumacetylsalicylate, dimenoxadol, dimepheptanol, dimethylthiambutene,dioxaphetyl butyrate, dipipanone, diprocetyl, dipyrone, ditazol,droxicam, emorfazone, enfenamic acid, enoxolone, epirizole, eptazocine,etersalate, ethenzamide, ethoheptazine, ethoxazene,ethylmethylthiambutene, ethylmorphine, etodolac, etofenamate,etonitazene, eugenol, felbinac, fenbufen, fenclozic acid, fendosal,fenoprofen, fentanyl, fentiazac, fepradinol, feprazone, floctafenine,fluazacort, flucloronide, flufenamic acid, flumethasone, flunisolide,flunixin, flunoxaprofen, fluocinolone acetonide, fluocinonide,fluocinolone acetonide, fluocortin butyl, fluocoitolone, fluoresone,fluorometholone, fluperolone, flupirtine, fluprednidene,fluprednisolone, fluproquazone, flurandrenolide, flurbiprofen,fluticasone, formocortal, fosfosal, gentisic acid, glafenine,glucametacin, glycol salicylate, guaiazulene, halcinonide, halobetasol,halometasone, haloprednone, heroin, hydrocodone, hydro cortamate,hydrocortisone, hydrocortisone acetate, hydrocortisone succinate,hydrocortisone hemisuccinate, hydrocortisone 21-lysinate, hydrocortisonecypionate, hydromorphone, hydroxypethidine, ibufenac, ibuprofen,ibuproxam, imidazole salicylate, indomethacin, indoprofen, isofezolac,isoflupredone, isoflupredone acetate, isoladol, isomethadone, isonixin,isoxepac, isoxicam, ketobemidone, ketoprofen, ketorolac,p-lactophenetide, lefetamine, levallorphan, levorphanol,levophenacyl-morphan, lofentanil, lonazolac, lornoxicam, loxoprofen,lysine acetylsalicylate, mazipredone, meclofenamic acid, medrysone,mefenamic acid, meloxicam, meperidine, meprednisone, meptazinol,mesalamine, metazocine, methadone, methotrimeprazine,methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, methylprednisolone suleptnate, metiazinic acid,metofoline, metopon, mofebutazone, mofezolac, mometasone, morazone,morphine, morphine hydrochloride, morphine sulfate, morpholinesalicylate, myrophine, nabumetone, nalbuphine, nalorphine, 1-naphthylsalicylate, naproxen, narceine, nefopam, nicomorphine, nifenazone,niflumic acid, nimesulide, 5′-nitro-2′-propoxyacetanilide,norlevorphanol, normethadone, normorphine, norpipanone, olsalazine,opium, oxaceprol, oxametacine, oxaprozin, oxycodone, oxymorphone,oxyphenbutazone, papaveretum, paramethasone, paranyline, parsalmide,pentazocine, perisoxal, phenacetin, phenadoxone, phenazocine,phenazopyridine hydrochloride, phenocoll, phenoperidine, phenopyrazone,phenomorphan, phenyl acetylsalicylate, phenylbutazone, phenylsalicylate, phenyramidol, piketoprofen, piminodine, pipebuzone,piperylone, pirazolac, piritramide, piroxicam, pirprofen, pranoprofen,prednicarbate, prednisolone, prednisone, prednival, prednylidene,proglumetacin, proheptazine, promedol, propacetamol, properidine,propiram, propoxyphene, propyphenazone, proquazone, protizinic acid,proxazole, ramifenazone, remifentanil, rimazolium metilsulfate,salacetamide, salicin, salicylamide, salicylamide o-acetic acid,salicylic acid, salicylsulfuric acid, salsalate, salverine, simetride,sufentanil, sulfasalazine, sulindac, superoxide dismutase, suprofen,suxibuzone, talniflumate, tenidap, tenoxicam, terofenamate, tetrandrine,thiazolinobutazone, tiaprofenic acid, tiaramide, tilidine, tinoridine,tixocortol, tolfenamic acid, tolmetin, tramadol, triamcinolone,triamcinolone acetonide, tropesin, viminol, xenbucin, ximoprofen,zaltoprofen and zomepirac.

In one embodiment, a compound described herein may be administered witha selective COX-2 inhibitor for treating or preventing inflammation.Exemplary selective COX-2 inhibitors include, for example, deracoxib,parecoxib, celecoxib, valdecoxib, rofecoxib, etoricoxib, andlumiracoxib.

In some embodiments, a provided compound is administered in combinationwith an anthracycline or a Topo II inhibitor. In certain embodiments, aprovided compound is administered in combination with Doxorubicin (Dox).In certain embodiments, a provided compound is administered incombination with bortezomib (and more broadly including carfilzomib). Itwas surprisingly found that a provided compound in combination with Doxor bortezomib resulted in a synergystic effect (i.e., more thanadditive).

Viral Infections

Compounds and methods described herein may be used to treat or prevent adisease or disorder associated with a viral infection, particularly inhumans and other mammals. A compound described herein may beadministered prior to the onset of, at, or after the initiation of viralinfection. When used prophylactically, the compounds are preferablyprovided in advance of any viral infection or symptom thereof.

Exemplary viral diseases include acute febrile pharyngitis,pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantilegastroenteritis, Coxsackie infections, infectious mononucleosis, Burkittlymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis,hepatocellular carcinoma, primary HSV-1 infection (e.g.,gingivostomatitis in children, tonsillitis and pharyngitis in adults,keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis andcold sores), primary HSV-2 infection, latent HSV-2 infection, asepticmeningitis, infectious mononucleosis, Cytomegalic inclusion disease,Kaposi's sarcoma, multicentric Castleman disease, primary effusionlymphoma, AIDS, influenza, Reye syndrome, measles, postinfectiousencephalomyelitis, Mumps, hyperplastic epithelial lesions (e.g., common,flat, plantar and anogenital warts, laryngeal papillomas,epidermodysplasia verruciformis), cervical carcinoma, squamous cellcarcinomas, croup, pneumonia, bronchiolitis, common cold, Poliomyelitis,Rabies, influenza-like syndrome, severe bronchiolitis with pneumonia,German measles, congenital rubella, Varicella, and herpes zoster.

Exemplary viral pathogens include Adenovirus, Coxsackievirus, Denguevirus, Encephalitis Virus, Epstein-Barr virus, Hepatitis A virus,Hepatitis B virus, Hepatitis C virus, Herpes simplex virus type 1,Herpes simplex virus type 2, cytomegalovirus, Human herpesvirus type 8,Human immunodeficiency virus, Influenza virus, measles virus, Mumpsvirus, Human papillomavirus, Parainfluenza virus, Poliovirus, Rabiesvirus, Respiratory syncytial virus, Rubella virus, Varicella-zostervirus, West Nile virus, Dungee, and Yellow fever virus. Viral pathogensmay also include viruses that cause resistant viral infections.

Antiviral drugs are a class of medications used specifically fortreating viral infections. Antiviral action generally falls into one ofthree mechanisms: interference with the ability of a virus to infiltratea target cell (e.g., amantadine, rimantadine and pleconaril), inhibitionof the synthesis of virus (e.g., nucleoside analogues, e.g., acyclovirand zidovudine (AZT), and inhibition of the release of virus (e.g.,zanamivir and oseltamivir).

Ophthalmology

Compounds and methods described herein may be used to treat or preventan ophthamology disorder. Exemplary ophthamology disorders includemacular edema (diabetic and nondiabetic macular edema), age relatedmacular degeneration wet and dry forms, aged disciform maculardegeneration, cystoid macular edema, palpebral edema, retina edema,diabetic retinopathy, chorioretinopathy, neovascular maculopathy,neovascular glaucoma, uveitis, iritis, retinal vasculitis,endophthalmitis, panophthalmitis, metastatic ophthalmia, choroiditis,retinal pigment epithelitis, conjunctivitis, cyclitis, scleritis,episcleritis, optic neuritis, retrobulbar optic neuritis, keratitis,blepharitis, exudative retinal detachment, corneal ulcer, conjunctivalulcer, chronic nummular keratitis, ophthalmic disease associated withhypoxia or ischemia, retinopathy of prematurity, proliferative diabeticretinopathy, polypoidal choroidal vasculopathy, retinal angiomatousproliferation, retinal artery occlusion, retinal vein occlusion, Coats'disease, familial exudative vitreoretinopathy, pulseless disease(Takayasu's disease), Eales disease, antiphospholipid antibody syndrome,leukemic retinopathy, blood hyperviscosity syndrome, macroglobulinemia,interferon-associated retinopathy, hypertensive retinopathy, radiationretinopathy, corneal epithelial stem cell deficiency and cataract.

Other ophthalmology disorders treatable using the compounds and methodsdescribed herein include proliferative vitreoretinopathy and chronicretinal detachment.

Inflammatory eye diseases are also treatable using the compounds andmethods described herein.

Neurodegenerative Disease

Neurodegeneration is the umbrella term for the progressive loss ofstructure or function of neurons, including death of neurons. Manyneurodegenerative diseases including Parkinson's, Alzheimer's, andHuntington's occur as a result of neurodegenerative processes. Asresearch progresses, many similarities appear which relate thesediseases to one another on a sub-cellular level. Discovering thesesimilarities offers hope for therapeutic advances that could amelioratemany diseases simultaneously. There are many parallels between differentneurodegenerative disorders including atypical protein assemblies aswell as induced cell death.

Alzheimer's disease is characterized by loss of neurons and synapses inthe cerebral cortex and certain subcortical regions. This loss resultsin gross atrophy of the affected regions, including degeneration in thetemporal lobe and parietal lobe, and parts of the frontal cortex andcingulate gyrus.

Huntington's disease causes astrogliosis and loss of medium spinyneurons. Areas of the brain are affected according to their structureand the types of neurons they contain, reducing in size as theycumulatively lose cells. The areas affected are mainly in the striatum,but also the frontal and temporal cortices. The striatum's subthalamicnuclei send control signals to the globus pallidus, which initiates andmodulates motion. The weaker signals from subthalamic nuclei thus causereduced initiation and modulation of movement, resulting in thecharacteristic movements of the disorder. Exemplary treatments forHuntington's disease include tetrabenazine, neuroleptics,benzodiazepines, amantadine, remacemide, valproic acid, selectiveserotonin reuptake inhibitors (SSRIs), mirtazapine and antipsychotics.

The mechanism by which the brain cells in Parkinson's are lost mayconsist of an abnormal accumulation of the protein alpha-synuclein boundto ubiquitin in the damaged cells. The alpha-synuclein-ubiquitin complexcannot be directed to the proteosome. This protein accumulation formsproteinaceous cytoplasmic inclusions called Lewy bodies. The latestresearch on pathogenesis of disease has shown that the death ofdopaminergic neurons by alpha-synuclein is due to a defect in themachinery that transports proteins between two major cellularorganelles—the endoplasmic reticulum (ER) and the Golgi apparatus.Certain proteins like Rab1 may reverse this defect caused byalpha-synuclein in animal models. Exemplary Parkinson's diseasetherapies include levodopa, dopamine agonists such as includebromocriptine, pergolide, pramipexole, ropinirole, piribedil,cabergoline, apomorphine and lisuride, dopa decarboxylate inhibitors,MAO-B inhibitors such as selegilene and rasagilene, anticholinergics andamantadine.

Amyotrophic lateral sclerosis (ALS/Lou Gehrig's Disease) is a disease inwhich motor neurons are selectively targeted for degeneration. ExemplaryALS therapies include riluzole, baclofen, diazepam, trihexyphenidyl andamitriptyline.

Other exemplary neurodegenerative therapeutics include antisenseoligonucleotides and stem cells.

Wound Healing

Wounds are a type of condition characterized by cell or tissue damage.Wound healing is a dynamic pathway that optimally leads to restorationof tissue integrity and function. The wound healing process consists ofthree overlapping phases. The first phase is an inflammatory phase,which is characterized by homeostasis, platelet aggregation anddegranulation. Platelets as the first response, release multiple growthfactors to recruit immune cells, epithelial cells, and endothelialcells. The inflammatory phase typically occurs over days 0-5. The secondstage of wound healing is the proliferative phase during whichmacrophages and granulocytes invade the wound. Infiltrating fibroblastsbegin to produce collagen. The principle characteristics of this phaseare epithelialization, angiogenesis, granulation tissue formation andcollagen production. The proliferative phase typically occurs over days3-14. The third phase is the remodeling phase where matrix formationoccurs. The fibroblasts, epithelial cells, and endothelial cellscontinue to produce collagen and collagenase as well as matrixmetalloproteases (MMPs) for remodeling. Collagen crosslinking takesplace and the wound undergoes contraction. The remodeling phasetypically occurs from day 7 to one year.

Compounds and compositions described herein can be used for promotingwound healing (e.g., promoting or accelerating wound closure and/orwound healing, mitigating scar fibrosis of the tissue of and/or aroundthe wound, inhibiting apoptosis of cells surrounding or proximate to thewound). Thus, in certain embodiments, the present invention provides amethod for promoting wound healing in a subject, comprisingadministering to the subject a compound (e.g., a CRM1 inhibitor), orpharmaceutically acceptable salt or composition thereof. The method neednot achieve complete healing or closure of the wound; it is sufficientfor the method to promote any degree of wound closure. In this respect,the method can be employed alone or as an adjunct to other methods forhealing wounded tissue.

The compounds and compositions described herein can be used to treatwounds during the inflammatory (or early) phase, during theproliferative (or middle) wound healing phase, and/or during theremodeling (or late) wound healing phase.

In some embodiments, the subject in need of wound healing is a human oran animal, for example, a horse, a pig, or a rodent, such as a mouse.

In some embodiments, the compounds and compositions described hereinuseful for wound healing are administered topically, for example,proximate to the wound site, or systemically.

More specifically, the compound or composition described herein can beadministered (optionally in combination with other agents) to the woundsite by coating the wound or applying a bandage, packing material,stitches, etc., that are coated or treated with the compound orcomposition described herein. As such, the compounds and compositionsdescribed herein can be formulated for topical administration to treatsurface wounds. Topical formulations include those for delivery via themouth (buccal) and to the skin such that a layer of skin (i.e., theepidermis, dermis, and/or subcutaneous layer) is contacted with thecompound or composition described herein. Topical delivery systems maybe used to administer topical formulations of the compounds andcompositions described herein.

Alternatively, the compounds and compositions described herein can beadministered at or near the wound site by, for example, injection of asolution, injection of an extended release formulation, or introductionof a biodegradable implant comprising the compound or compositiondescribed herein.

The compounds and compositions described herein can be used to treatacute wounds or chronic wounds. A chronic wound results when the normalreparative process is interrupted. Chronic wounds can develop from acuteinjuries as a result of unrecognized persistent infections or inadequateprimary treatment. In most cases however, chronic lesions are the endstage of progressive tissue breakdown owing to venous, arterial, ormetabolic vascular disease, pressure sores, radiation damage, or tumors.

In chronic wounds, healing does not occur for a variety of reasons,including improper circulation in diabetic ulcers, significant necrosis,such as in burns, and infections. In these chronic wounds, viability orthe recovery phase is often the rate-limiting step. The cells are nolonger viable and thus initial recovery phase is prolonged byunfavorable wound bed environment.

Chronic wounds include, but are not limited to the following: chronicischemic skin lesions; scleroderma ulcers; arterial ulcers; diabeticfoot ulcers; pressure ulcers; venous ulcers; non-healing lower extremitywounds; ulcers due to inflammatory conditions; and/or long-standingwounds.

In a particular embodiment, the compounds and compositions describedherein can be used for diabetic wound healing or accelerating healing ofleg and foot ulcers secondary to diabetes or ischemia in a subject.

In one embodiment, the wound is a surface wound. In another embodiment,the wound is a surgical wound (e.g., abdominal or gastrointestinalsurgical wound). In a further embodiment, the wound is a burn. In yetanother embodiment, the wound is the result of radiation exposure.

The compounds and compositions described herein can also be used fordiabetic wound healing, gastrointestinal wound healing, or healing of anadhesion due, for example, to an operation.

The compounds and compositions described herein can also be used to healwounds that are secondary to another disease. For example, ininflammatory skin diseases, such as psoriasis and dermatitis, there arenumerous incidents of skin lesions that are secondary to the disease,and are caused by deep cracking of the skin, or scratching of the skin.The compounds and compositions described herein can be used to healwounds that are secondary to these diseases, for example, inflammatoryskin diseases, such as psoriasis and dermatitis.

In a further embodiment, the wound is an internal wound. In a specificaspect, the internal wound is a chronic wound. In another specificaspect, the wound is a vascular wound. In yet another specific aspect,the internal wound is an ulcer.

Examples of wounds include, but are not limited to, abrasions,avulsions, blowing wounds (i.e., open pneumothorax), burn wounds,contusions, gunshot wounds, incised wounds, open wounds, penetratingwounds, perforating wounds, puncture wounds, séton wounds, stab wounds,surgical wounds, subcutaneous wounds, diabetic lesions, or tangentialwounds. Additional examples of wounds that can be treated by thecompounds and compositions described herein include acute conditions orwounds, such as thermal burns, chemical burns, radiation burns, burnscaused by excess exposure to ultraviolet radiation (e.g., sunburn);damage to bodily tissues, such as the perineum as a result of labor andchildbirth; injuries sustained during medical procedures, such asepisiotomies; trauma-induced injuries including cuts, incisions,excoriations; injuries sustained from accidents; post-surgical injuries,as well as chronic conditions, such as pressure sores, bedsores,conditions related to diabetes and poor circulation, and all types ofacne. In addition, the wound can include dermatitis, such as impetigo,intertrigo, folliculitis and eczema, wounds following dental surgery;periodontal disease; wounds following trauma; and tumor-associatedwounds. Yet other examples of wounds include animal bites, arterialdisease, insect stings and bites, bone infections, compromisedskin/muscle grafts, gangrene, skin tears or lacerations, skin aging,surgical incisions, including slow or non-healing surgical wounds,intracerebral hemorrhage, aneurysm, dermal asthenia, and post-operationinfections.

In preferred embodiments, the wound is selected from the groupconsisting of a burn wound, an incised wound, an open wound, a surgicalor post surgical wound, a diabetic lesion, a thermal burn, a chemicalburn, a radiation burn, a pressure sore, a bedsore, and a conditionrelated to diabetes or poor circulation.

The present disclosure also relates to methods and compositions ofreducing scar formation during wound healing in a subject. The compoundsand compositions described herein can be administered directly to thewound or to cells proximate the wound at an amount effective to reducescar formation in and/or around the wound.

The wound can include any injury to any portion of the body of asubject. According to embodiments, methods are provided to ameliorate,reduce, or decrease the formation of scars in a subject that hassuffered a burn injury. According to preferred embodiments, methods areprovided to treat, reduce the occurrence of, or reduce the probabilityof developing hypertrophic scars in a subject that has suffered an acuteor chronic wound or injury.

Other Disorders

Compounds and compositions described herein may also be used to treatdisorders of abnormal tissue growth and fibrosis including dilativecardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,pulmonary fibrosis, hepatic fibrosis, glomerulonephritis, and otherrenal disorders.

Combination Radiation Therapy

Compounds and compositions described herein are useful asradiosensitizers. Therefore, compounds and compositions described hereincan be administered in combination with radiation therapy. Radiationtherapy is the medical use of high-energy radiation (e.g., x-rays, gammarays, charged particles) to shrink tumors and kill malignant cells, andis generally used as part of cancer treatment. Radiation therapy killsmalignant cells by damaging their DNA.

Radiation therapy can be delivered to a patient in several ways. Forexample, radiation can be delivered from an external source, such as amachine outside the patient's body, as in external beam radiationtherapy. External beam radiation therapy for the treatment of canceruses a radiation source that is external to the patient, typicallyeither a radioisotope, such as ⁶⁰Co, ¹³⁷Cs, or a high energy x-raysource, such as a linear accelerator. The external source produces acollimated beam directed into the patient to the tumor site.External-source radiation therapy avoids some of the problems ofinternal-source radiation therapy, but it undesirably and necessarilyirradiates a significant volume of non-tumorous or healthy tissue in thepath of the radiation beam along with the tumorous tissue.

The adverse effect of irradiating of healthy tissue can be reduced,while maintaining a given dose of radiation in the tumorous tissue, byprojecting the external radiation beam into the patient at a variety of“gantry” angles with the beams converging on the tumor site. Theparticular volume elements of healthy tissue, along the path of theradiation beam, change, reducing the total dose to each such element ofhealthy tissue during the entire treatment.

The irradiation of healthy tissue also can be reduced by tightlycollimating the radiation beam to the general cross section of the tumortaken perpendicular to the axis of the radiation beam. Numerous systemsexist for producing such a circumferential collimation, some of whichuse multiple sliding shutters which, piecewise, can generate aradio-opaque mask of arbitrary outline.

For administration of external beam radiation, the amount can be atleast about 1 Gray (Gy) fractions at least once every other day to atreatment volume. In a particular embodiment, the radiation isadministered in at least about 2 Gray (Gy) fractions at least once perday to a treatment volume. In another particular embodiment, theradiation is administered in at least about 2 Gray (Gy) fractions atleast once per day to a treatment volume for five consecutive days perweek. In another particular embodiment, radiation is administered in 10Gy fractions every other day, three times per week to a treatmentvolume. In another particular embodiment, a total of at least about 20Gy is administered to a patient in need thereof. In another particularembodiment, at least about 30 Gy is administered to a patient in needthereof. In another particular embodiment, at least about 40 Gy isadministered to a patient in need thereof.

Typically, the patient receives external beam therapy four or five timesa week. An entire course of treatment usually lasts from one to sevenweeks depending on the type of cancer and the goal of treatment. Forexample, a patient can receive a dose of 2 Gy/day over 30 days.

Internal radiation therapy is localized radiation therapy, meaning theradiation source is placed at the site of the tumor or affected area.Internal radiation therapy can be delivered by placing a radiationsource inside or next to the area requiring treatment. Internalradiation therapy is also called brachytherapy. Brachytherapy includesintercavitary treatment and interstitial treatment. In intracavitarytreatment, containers that hold radioactive sources are put in or nearthe tumor. The sources are put into the body cavities. In interstitialtreatment, the radioactive sources alone are put into the tumor. Theseradioactive sources can stay in the patient permanently. Typically, theradioactive sources are removed from the patient after several days. Theradioactive sources are in containers.

There are a number of methods for administration of aradiopharmaceutical agent. For example, the radiopharmaceutical agentcan be administered by targeted delivery or by systemic delivery oftargeted radioactive conjugates, such as a radiolabeled antibody, aradiolabeled peptide and a liposome delivery system. In one particularembodiment of targeted delivery, the radiolabelled pharmaceutical agentcan be a radiolabelled antibody. See, for example, Ballangrud A. M., etal. Cancer Res., 2001; 61:2008-2014 and Goldenber, D. M. J. Nucl. Med.,2002; 43(5):693-713, the contents of which are incorporated by referenceherein.

In another particular embodiment of targeted delivery, theradiopharmaceutical agent can be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines. See, for example, Emfietzoglou D, Kostarelos K,Sgouros G. An analytical dosimetry study for the use ofradionuclide-liposome conjugates in internal radiotherapy. J Nucl Med2001; 42:499-504, the contents of which are incorporated by referenceherein.

In yet another particular embodiment of targeted delivery, theradiolabeled pharmaceutical agent can be a radiolabeled peptide. See,for example, Weiner R E, Thakur M L. Radiolabeled peptides in thediagnosis and therapy of oncological diseases. Appl Radiat Isot 2002November; 57(5):749-63, the contents of which are incorporated byreference herein.

In addition to targeted delivery, bracytherapy can be used to deliverthe radiopharmaceutical agent to the target site. Brachytherapy is atechnique that puts the radiation sources as close as possible to thetumor site. Often the source is inserted directly into the tumor. Theradioactive sources can be in the form of wires, seeds or rods.Generally, cesium, iridium or iodine are used.

Systemic radiation therapy is another type of radiation therapy andinvolves the use of radioactive substances in the blood. Systemicradiation therapy is a form of targeted therapy. In systemic radiationtherapy, a patient typically ingests or receives an injection of aradioactive substance, such as radioactive iodine or a radioactivesubstance bound to a monoclonal antibody.

A “radiopharmaceutical agent,” as defined herein, refers to apharmaceutical agent which contains at least one radiation-emittingradioisotope. Radiopharmaceutical agents are routinely used in nuclearmedicine for the diagnosis and/or therapy of various diseases. Theradiolabelled pharmaceutical agent, for example, a radiolabelledantibody, contains a radioisotope (RI) which serves as the radiationsource. As contemplated herein, the term “radioisotope” includesmetallic and non-metallic radioisotopes. The radioisotope is chosenbased on the medical application of the radiolabeled pharmaceuticalagents. When the radioisotope is a metallic radioisotope, a chelator istypically employed to bind the metallic radioisotope to the rest of themolecule. When the radioisotope is a non-metallic radioisotope, thenon-metallic radioisotope is typically linked directly, or via a linker,to the rest of the molecule.

As used herein, a “metallic radioisotope” is any suitable metallicradioisotope useful in a therapeutic or diagnostic procedure in vivo orin vitro. Suitable metallic radioisotopes include, but are not limitedto: Actinium-225, Antimony-124, Antimony-125, Arsenic-74, Barium-103,Barium-140, Beryllium-7, Bismuth-206, Bismuth-207, Bismuth212,Bismuth213, Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139,Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-55, Cobalt-56,Cobalt-57, Cobalt-58, Cobalt-60, Cobalt-64, Copper-60, Copper-62,Copper-64, Copper-67, Erbium-169, Europium-152, Gallium-64, Gallium-67,Gallium-68, Gadolinium 153, Gadolinium-157 Gold-195, Gold-199,Hafnium-175, Hafnium-175-181, Holmium-166, Indium-110, Indium-111,Iridium-192, Iron 55, Iron-59, Krypton85, Lead-203, Lead-210,Lutetium-177, Manganese-54, Mercury-197, Mercury203, Molybdenum-99,Neodymium-147, Neptunium-237, Nickel-63, Niobium95, Osmium-185+191,Palladium-103, Palladium-109, Platinum-195m, Praseodymium-143,Promethium-147, Promethium-149, Protactinium-233, Radium-226,Rhenium-186, Rhenium-188, Rubidium-86, Ruthenium-97, Ruthenium-103,Ruthenium-105, Ruthenium-106, Samarium-153, Scandium-44, Scandium-46,Scandium-47, Selenium-75, Silver-110m, Silver-111, Sodium-22,Strontium-85, Strontium-89, Strontium-90, Sulfur-35, Tantalum-182,Technetium-99m, Tellurium-125, Tellurium-132, Thallium-204, Thorium-228,Thorium-232, Thallium-170, Tin-113, Tin-114, Tin-117m, Titanium-44,Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169, Yttrium-86,Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, Zirconium-89, andZirconium-95.

As used herein, a “non-metallic radioisotope” is any suitablenonmetallic radioisotope (non-metallic radioisotope) useful in atherapeutic or diagnostic procedure in vivo or in vitro. Suitablenon-metallic radioisotopes include, but are not limited to: Iodine-131,Iodine-125, Iodine-123, Phosphorus-32, Astatine-211, Fluorine-18,Carbon-11, Oxygen-15, Bromine-76, and Nitrogen-13.

Identifying the most appropriate isotope for radiotherapy requiresweighing a variety of factors. These include tumor uptake and retention,blood clearance, rate of radiation delivery, half-life and specificactivity of the radioisotope, and the feasibility of large-scaleproduction of the radioisotope in an economical fashion. The key pointfor a therapeutic radiopharmaceutical is to deliver the requisite amountof radiation dose to the tumor cells and to achieve a cytotoxic ortumoricidal effect while not causing unmanageable side-effects.

It is preferred that the physical half-life of the therapeuticradioisotope be similar to the biological half-life of theradiopharmaceutical at the tumor site. For example, if the half-life ofthe radioisotope is too short, much of the decay will have occurredbefore the radiopharmaceutical has reached maximum target/backgroundratio. On the other hand, too long a half-life could cause unnecessaryradiation dose to normal tissues. Ideally, the radioisotope should havea long enough half-life to attain a minimum dose rate and to irradiateall the cells during the most radiation sensitive phases of the cellcycle. In addition, the half-life of a radioisotope has to be longenough to allow adequate time for manufacturing, release, andtransportation.

Other practical considerations in selecting a radioisotope for a givenapplication in tumor therapy are availability and quality. The purityhas to be sufficient and reproducible, as trace amounts of impuritiescan affect the radiolabeling and radiochemical purity of theradiopharmaceutical.

The target receptor sites in tumors are typically limited in number. Assuch, it is preferred that the radioisotope have high specific activity.The specific activity depends primarily on the production method. Tracemetal contaminants must be minimized as they often compete with theradioisotope for the chelator and their metal complexes compete forreceptor binding with the radiolabeled chelated agent.

The type of radiation that is suitable for use in the methods of thepresent invention can vary. For example, radiation can beelectromagnetic or particulate in nature. Electromagnetic radiationuseful in the practice of this invention includes, but is not limitedto, x-rays and gamma rays. Particulate radiation useful in the practiceof this invention includes, but is not limited to, electron beams (betaparticles), protons beams, neutron beams, alpha particles, and negativepi mesons. The radiation can be delivered using conventionalradiological treatment apparatus and methods, and by intraoperative andstereotactic methods. Additional discussion regarding radiationtreatments suitable for use in the practice of this invention can befound throughout Steven A. Leibel et al., Textbook of Radiation Oncology(1998) (publ. W. B. Saunders Company), and particularly in Chapters 13and 14. Radiation can also be delivered by other methods such astargeted delivery, for example by radioactive “seeds,” or by systemicdelivery of targeted radioactive conjugates. J. Padawer et al., CombinedTreatment with Radioestradiol lucanthone in Mouse C3HBA MammaryAdenocarcinoma and with Estradiol lucanthone in an Estrogen Bioassay,Int. J. Radiat. Oncol. Biol. Phys. 7:347-357 (1981). Other radiationdelivery methods can be used in the practice of this invention.

For tumor therapy, both α and β-particle emitters have beeninvestigated. Alpha particles are particularly good cytotoxic agentsbecause they dissipate a large amount of energy within one or two celldiameters. The β-particle emitters have relatively long penetrationrange (2-12 mm in the tissue) depending on the energy level. Thelong-range penetration is particularly important for solid tumors thathave heterogeneous blood flow and/or receptor expression. The β-particleemitters yield a more homogeneous dose distribution even when they areheterogeneously distributed within the target tissue.

In a particular embodiment, therapeutically effective amounts of thecompounds and compositions described herein are administered incombination with a therapeutically effective amount of radiation therapyto treat cancer (e.g., lung cancer, such as non-small cell lung cancer).The amount of radiation necessary can be determined by one of skill inthe art based on known doses for a particular type of cancer. See, forexample, Cancer Medicine 5^(th) ed., Edited by R. C. Bast et al., July2000, BC Decker.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXEMPLIFICATION Abbreviations

aq. AqueousBoc tert-butoxycarbonylCH₂Cl₂ DichloromethaneDABCO 1,4-diazabicyclo[2.2.2]octane

DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide DMSODimethylsulfoxide

eq. equivalent(s)EtOAc Ethyl acetate

EtOH Ethanol

h hour(s)HPLC High performance liquid chromatography

LCMS Liquid Chromatography Mass Spectrometry

LiOH Lithium hydroxideNMR Nuclear magnetic resonance

RT Room Temperature or Retention Time

T3P Propylphosphonic anhydrideTFA Trifluoroacetic acid

THF Tetrahydrofuran

Throughout the following description of such processes it is to beunderstood that, where appropriate, suitable protecting groups will beadded to, and subsequently removed from, the various reactants andintermediates in a manner that will be readily understood by one skilledin the art of organic synthesis. Conventional procedures for using suchprotecting groups as well as examples of suitable protecting groups aredescribed, for example, in “Protective Groups in Organic Synthesis”, T.W. Green, P. G. M. Wuts, Wiley-Interscience, New York, (1999). It isalso to be understood that a transformation of a group or substituentinto another group or substituent by chemical manipulation can beconducted on any intermediate or final product on the synthetic pathtoward the final product, in which the possible type of transformationis limited only by inherent incompatibility of other functionalitiescarried by the molecule at that stage to the conditions or reagentsemployed in the transformation. Such inherent incompatibilities, andways to circumvent them by carrying out appropriate transformations andsynthetic steps in a suitable order, will be readily understood to theone skilled in the art of organic synthesis. Examples of transformationsare given below, and it is to be understood that the describedtransformations are not limited only to the generic groups orsubstituents for which the transformations are exemplified. Referencesand descriptions on other suitable transformations are given in“Comprehensive Organic Transformations—A Guide to Functional GroupPreparations” R. C. Larock, VHC Publishers, Inc. (1989). References anddescriptions of other suitable reactions are described in textbooks oforganic chemistry, for example, “Advanced Organic Chemistry”, March, 4thed. McGraw Hill (1992) or, “Organic Synthesis”, Smith, McGraw Hill,(1994). Techniques for purification of intermediates and final productsinclude for example, straight and reversed phase chromatography oncolumn or rotating plate, recrystallization, distillation andliquid-liquid or solid-liquid extraction, which will be readilyunderstood by the one skilled in the art. The definitions ofsubstituents and groups are as in formula I except where defineddifferently. The term “room temperature” and “ambient temperature” shallmean, unless otherwise specified, a temperature between 16 and 25° C.The term “reflux” shall mean, unless otherwise stated, in reference toan employed solvent a temperature at or above the boiling point of namedsolvent.

Example 1. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-pivaloylacrylohydrazide(Compound 1). Compound 1 was Synthesized According to the FollowingScheme

3,5-bis(trifluoromethyl)benzothioamide (Step 1)

A 2 L 3-neck round-bottom flask was charged with a solution of3,5-bis(trifluoromethyl)benzonitrile (200 g) in DMF (1 L). The solutionwas then treated with NaSH (123.7 g, 2.0 eq.) and MgCl₂ (186.7 g, 1.0eq.) and the reaction mixture was stirred at RT for 3 h. The mixture waspoured into an ice-water slurry (10 L) and the compound was extractedwith EtOAc (3×1 L). The combined organic layers were washed with aqueoussaturated sodium chloride solution (3×100 mL), dried over anhydrousNa₂SO₄, filtered, and concentrated under reduced pressure to afford 205g of the desired crude 3,5-bis(trifluoromethyl)benzothioamide (yield:90%), which was used without further purification in the following step.

3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole (Step 2)

A 5 L 3-neck round-bottom flask was charged with a solution of3,5-bis(trifluoromethyl)benzothioamide (205.65 g) in DMF (1.03 L).Hydrazine hydrate (73.2 mL, 2.0 eq.) was added dropwise and the reactionmixture was stirred at RT for 1 h. HCOOH (1.03 L) was added dropwise andthe reaction mixture was refluxed at 90° C. for 3 h. After being allowedto cool down to RT, the reaction mixture was poured into saturatedaqueous sodium bicarbonate solution (7 L) and extracted with EtOAc (3×1L). The combined organic layers were washed with aqueous saturatedsodium chloride solution (3×500 mL), dried over anhydrous Na₂SO₄,filtered, and concentrated under reduced pressure (35° C., 20 mmHg) toafford 180 g of the crude product. The crude material was stirred withpetroleum ether (3×500 mL), filtered, and dried to obtain 160 g of thedesired 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole obtained asa pale yellow solid (yield: 75%).

(Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate(Step 3)

A 2 L 3-neck round-bottom flask was charged with a solution of3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole (160 g) in DMF (960mL). The solution was treated with DABCO (127.74 g, 2 eq.) and stirredfor 30 min before adding (Z)-isopropyl 3-iodoacrylate (150.32 g, 1.1eq.) dropwise. After 1 h, the reaction mixture was poured into anice-water slurry (5 L) and extracted with EtOAc (3×1 L). The combinedorganic layers were washed with aqueous saturated sodium chloridesolution (3×100 mL), dried over anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure (35° C., 20 mmHg) to afford 250 g ofthe crude product that was purified by column chromatography (60/120silica gel) using ethyl acetate/n-hexane gradient (the column was packedin hexane and the desired compound started eluting from 2%EtOAC/n-hexane). Fractions containing the desired compounds werecombined to afford (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate(138 g, yield: 61%).

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Step 4)

In a 5 L, 3-neck round-bottom flask, (Z)-isopropyl3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate(130 g, 1.0 eq.) was dissolved in THF (1.3 L). A solution of LiOH (69.3g, 5.0 eq.) in water (1.3 L) was added dropwise to the solution and thereaction mixture was stirred at RT for 4 h before being quenched with400 mL ice-water slurry and made acidic (pH=2-3) with dilute aqueousHCl. The mixture was extracted with EtOAc (3×1 L) and the combinedorganic layers were washed with aqueous saturated sodium chloridesolution, dried over anhydrous Na₂SO₄, and concentrated under reducedpressure to afford 110 g of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (yield: 94%), (cis content=90.0%, trans content=8.2% by LCMS).

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-pivaloylacrylohydrazide(Compound 1)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.2 g, 1.0 eq.) was dissolved in EtOAc (20 mL) and cooled to −60°C. where pivalohydrazide (0.08 g, 1.2 eq.) was introduced dropwise. T3P(50% in EtOAc) (0.4 mL, 4 eq.) was added dropwise followed by DIPEA (0.4mL, 4 eq.) and the reaction mixture was stirred for 1 h at −60° C. Thereaction mixture was concentrated under reduced pressure (25° C., 20 mmHg) to afford the crude product that was purified by columnchromatography (60/120 silica gel) using methanol/dichloromethanegradient (the column was packed in dichloromethane and the desiredcompound started eluting from 3% methanol/dichloromethane). Fractionscontaining the desired compounds were combined to afford(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-pivaloylacrylohydrazide(0.11 g, yield: 43%);

Example 2. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-morpholinoacetyl)acrylohydrazide(Compound 2) 2-morpholinoacetohydrazide

In a 25 mL, 3-neck round-bottom flask, methyl 2-morpholinoacetate (0.25g, 1.0 eq.) was dissolved in ethanol (5 mL) at RT. Hydrazine hydrate(0.087 g, 1.1 eq.) was introduced dropwise at RT and the reactionmixture was refluxed at 95° C. for 20 h. The reaction mixture wasconcentrated under reduced pressure (40° C., 20 mm Hg) to afford thecrude 2-morpholinoacetohydrazide (0.23 g) which was used without furtherpurification in the following step.

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-morpholinoacetyl)acrylohydrazide(Compound 2)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Example 1, Step 4; 0.5 g, 1.0 eq.) was dissolved in CH₂Cl₂: EtOAc(20 mL, 2:1) and cooled to −60° C. where 2-morpholinoacetohydrazide(0.23 g, 1.0 eq.) was introduced dropwise. T3P (50% in EtOAc) (1.27 mL,1.5 eq.) was added dropwise followed by DIPEA (0.96 mL, 2 eq.) and thereaction mixture was stirred for 1 h at −60° C. The reaction mixture wasconcentrated under reduced pressure (25° C., 20 mm Hg) to afford thecrude product that was purified by column chromatography (60/120 silicagel) using methanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-morpholinoacetyl)acrylohydrazide(0.1 g, yield: 14%).

Example 3. Synthesis of(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-5-methyl-1H-pyrazole-4-carbohydrazide(Compound 3) 5-methyl-1H-pyrazole-4-carbohydrazide

In a 25 mL sealed tube, ethyl 5-methyl-1H-pyrazole-4-carboxylate (0.25g, 1.0 eq.) was dissolved in ethanol (5 mL) at RT. Hydrazine hydrate (1mL, 5 eq.) was introduced dropwise at RT and the reaction mixture washeated at 120° C. for 20 h. The reaction mixture was concentrated underreduced pressure (40° C., 20 mm Hg) to afford the crude5-methyl-1H-pyrazole-4-carbohydrazide (0.24 g) which was used withoutfurther purification in the following step.

(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-5-methyl-1H-pyrazole-4-carbohydrazide(Compound 3)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Example 1, Step 4; 0.5 g, 1.0 eq.) was dissolved in EtOAc:EtOH (15mL, 2:1) and cooled to −60° C. where5-methyl-1H-pyrazole-4-carbohydrazide (0.24 g, 1.0 eq.) was introduceddropwise. T3P (50% in EtOAc) (1.69 mL, 1.5 eq.) was added dropwisefollowed by DIPEA (2 mL, 8 eq.) and the reaction mixture was stirred for1 h at −60° C. The reaction mixture was concentrated under reducedpressure (25° C., 20 mm Hg) to afford the crude product that waspurified by column chromatography (60/120 silica gel) usingmethanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-5-methyl-1H-pyrazole-4-carbohydrazide(0.2 g, yield: 42%).

Example 4. Synthesis of(Z)-2-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-N-cyclopropylhydrazinecarbothioamide(Compound 4)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Example 1, Step 4; 0.5 g, 1.0 eq.) was dissolved in EtOAc:EtOH (15mL, 2:1) and cooled to −60° C. whereN-cyclopropylhydrazinecarbothioamide (0.22 g, 1.2 eq.) was introduceddropwise. T3P (50% in EtOAc) (1.69 mL, 2 eq.) was added dropwisefollowed by DIPEA (1 mL, 4 eq.) and the reaction mixture was stirred for1 h at −60° C. The reaction mixture was concentrated under reducedpressure (25° C., 20 mm Hg) to afford the crude product that waspurified by column chromatography (60/120 silica gel) usingmethanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(Z)-2-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-N-cyclopropylhydrazinecarbothioamide(0.06 g, yield: 9%).

Example 5. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(2-morpholinoacetyl)acrylohydrazide(Compound 5) N-methyl-2-morpholinoacetohydrazide

In a 25 mL, sealed tube, methyl 2-morpholinoacetate (0.5 g, 1.0 eq.) wasdissolved in ethanol (5 mL) at RT. Methylhydrazine (0.16 g, 1.1 eq.) wasintroduced dropwise at RT and the reaction mixture was refluxed at 95°C. for 48 h. The reaction mixture was concentrated under reducedpressure (40° C., 20 mm Hg) to afford the crudeN-methyl-2-morpholinoacetohydrazide (0.27 g) which was used withoutfurther purification in the following step.

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(2-morpholinoacetyl)acrylohydrazide(Compound 5)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Example 1, Step 4; 0.3 g, 1.0 eq.) was dissolved in THF:EtOAc (15mL, 2:1) and cooled to −60° C. where N-methyl-2-morpholinoacetohydrazide(0.23 g, 1.5 eq.) was introduced dropwise. T3P (50% in EtOAc) (1.27 mL,2.5 eq.) was added dropwise followed by DIPEA (0.45 mL, 3 eq.) and thereaction mixture was stirred for 1 h at −60° C. The reaction mixture wasconcentrated under reduced pressure (25° C., 20 mm Hg) to afford thecrude product that was purified by column chromatography (60/120 silicagel) using methanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-methyl-N′-(2-morpholinoacetyl)acrylohydrazide(0.052 g, yield: 12%).

Example 6. Synthesis of(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)piperidine-3-carbohydrazide(Compound 6) Piperidine-3-carbohydrazide

In a 30 mL sealed tube, ethyl methyl piperidine-3-carboxylate (1 g, 1.0eq.) was dissolved in ethanol (5 mL) at RT. Hydrazine hydrate (1.05 g, 3eq.) was introduced dropwise at RT and the reaction mixture was heatedat 120° C. for 20 h. The reaction mixture was concentrated under reducedpressure (40° C., 20 mm Hg) to afford the crudepiperidine-3-carbohydrazide (0.8 g) which was used without furtherpurification in the following step.

(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)piperidine-3-carbohydrazide(Compound 6)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Example 1, Step 4; 0.25 g, 1.0 eq.) was dissolved in THF:EtOAc (15mL, 2:1) and cooled to −60° C. where piperidine-3-carbohydrazide (0.113g, 1.1 eq.) was introduced dropwise. T3P (50% in EtOAc) (1.69 mL, 4 eq.)was added dropwise followed by DIPEA (0.25 mL, 2 eq.) and the reactionmixture was stirred for 1 h at −60° C. The reaction mixture wasconcentrated under reduced pressure (25° C., 20 mm Hg) to afford thecrude product that was purified by column chromatography (60/120 silicagel) using methanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)piperidine-3-carbohydrazide(0.01 g, yield: 2.4%).

Example 7. Synthesis of(S,Z)-2-amino-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-3-methylbutanehydrazide2,2,2-trifluoroacetate (Compound 7)

Compound 7 was synthesized by the following scheme:

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylohydrazide(Step 1)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Example 1, Step 4; 0.5 g, 1.0 eq.) was dissolved in THF (10 mL)and cooled to −10° C. where NMP (0.3 g, 2.1 eq.) was added and thereaction mixture was stirred for 5 min. Isobutyl chloroformate (0.465 g,2.4 eq.) was then added and the reaction mixture was stirred for 1 h.The solid formed was removed by filtration. The filtrate was cooled to0° C. and tert-butoxycarbonyl hydrazide (0.21 g, 1.1 eq.) wasintroduced. The reaction mixture was allowed to warm to RT where it wasstirred for 1 h. The reaction mixture was poured into an iced-waterslurry and extracted with EtOAc (3×50 mL). The combined organic layerswere washed with aqueous saturated sodium chloride solution (25 mL),dried over anhydrous Na₂SO₄, filtered, and concentrated under reducedpressure (25° C., 20 mmHg) to afford 0.5 g of the crude product. Thecrude product was then dissolved in THF (10 mL) and TFA (2 mL) was addeddropwise at RT and the reaction mixture was stirred for 2 h. Thereaction mixture was concentrated under reduced pressure (25° C., 20mmHg) and the solid formed was triturated with pentane to afford(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylohydrazide(0.25 g, yield: 48.5%).

(S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid

In a 25 mL, 3-neck round-bottom flask, (S)-2-amino-3-methylbutanoic acid(0.8 g, 1.0 eq.) was dissolved in water (4 mL). Sodium bicarbonate (0.63g, 1.1 eq.), followed by di-tert-butyl dicarbonate (2.97 g, 2.0 eq.) wasadded and the reaction mixture was stirred for 2 h at RT. The reactionmixture was extracted with EtOAc (3×10 mL). The combined organic layerswere washed with aqueous saturated sodium chloride solution (25 mL),dried over anhydrous Na₂SO₄, filtered, and concentrated under reducedpressure (25° C., 20 mmHg) to afford 1.2 g of the crude product that waspurified by column chromatography (60/120 silica gel) usingmethanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (0.7 g, yield:47.3%).

(S,Z)-2-amino-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-3-methylbutanehydrazide2,2,2-trifluoroacetate (Compound 7)

In a 10 mL round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylohydrazide(0.25 g, 1.0 eq.) was dissolved in THF (5 mL) and cooled to −60° C.where (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (0.19 g,1.3 eq.) was introduced dropwise. T3P (50% in EtOAc) (0.81 mL, 2 eq.)was added dropwise followed by DIPEA (0.48 mL, 4 eq.) and the reactionmixture was stirred for 1 h at −60° C. The reaction mixture wasconcentrated under reduced pressure (25° C., 20 mm Hg) to afford thecrude product that was purified by column chromatography (60/120 silicagel) using methanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford (S,Z)-tert-butyl(1-(2-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)hydrazinyl)-3-methyl-1-oxobutan-2-yl)carbamate(0.07 g, yield: 18%). In a 10 mL round-bottom flask, (S,Z)-tert-butyl(1-(2-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)hydrazinyl)-3-methyl-1-oxobutan-2-yl)carbamatewas then dissolved in dichloromethane (2 mL). TFA (0.05 mL) was addedand the reaction mixture was stirred at RT for 5 h. The reaction mixturewas concentrated under reduced pressure (25° C., 20 mm Hg) to afford thecrude product (0.01 g), which was triturated with petroleum ether anddried under reduced pressure to yield(S,Z)-2-amino-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-3-methylbutanehydrazide2,2,2-trifluoroacetate (0.006 g, yield: 2%).

Example 8. Synthesis of(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)pyrazine-2-carbohydrazide(Compound 8)

In a 25 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (Example 1, Step 4; 0.5 g, 1.0 eq.) was dissolved indichloromethane (5 mL) and cooled to −60° C. wherepyrazine-2-carbohydrazide (0.216 g, 1.1 eq.) was introduced. T3P (50% inEtOAc) (3.39 mL, 4 eq.) was added dropwise followed by DIPEA (0.5 mL, 2eq.) and the reaction mixture was stirred for 1 h at −60° C. Thereaction mixture was concentrated under reduced pressure (25° C., 20 mmHg) to afford the crude product that was purified by columnchromatography (60/120 silica gel) using methanol/dichloromethanegradient (the column was packed in dichloromethane and the desiredcompound started eluting from 3% methanol/dichloromethane). Fractionscontaining the desired compounds were combined to afford(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)pyrazine-2-carbohydrazide(0.13 g, yield: 19.4%).

Example 9. Synthesis of(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-1-methylpiperidine-4-carbohydrazide(Compound 9) 1-methylpiperidine-4-carbohydrazide

In a 25 mL sealed tube, methyl 1-methylpiperidine-4-carboxylate (0.2 g,1.0 eq.) was dissolved in ethanol (5 mL) at RT. Hydrazine hydrate (0.127g, 2 eq.) was introduced dropwise at RT and the reaction mixture washeated at 120° C. for 20 h. The reaction mixture was concentrated underreduced pressure (40° C., 20 mm Hg) to afford the crude1-methylpiperidine-4-carbohydrazide (0.145 g) which was used withoutfurther purification in the following step.

(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-1-methylpiperidine-4-carbohydrazide(Compound 9)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.25 g, 1.0 eq.) was dissolved in EtOAc:THF (15 mL; 2:1) andcooled to −60° C. where 1-methylpiperidine-4-carbohydrazide (0.123 g,1.1 eq.) was introduced. T3P (50% in EtOAc) (0.85 mL, 2 eq.) was addeddropwise followed by DIPEA (0.31 mL, 2.5 eq.) and the reaction mixturewas stirred for 1 h at −60° C. The reaction mixture was concentratedunder reduced pressure (35° C., 20 mm Hg) to afford the crude productthat was purified by column chromatography (60/120 silica gel) usingmethanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(Z)-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-1-methylpiperidine-4-carbohydrazide(0.016 g, yield: 4.5%).

Example 10. Synthesis of(R,Z)-2-amino-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-3-methylbutanehydrazide2,2,2-trifluoroacetate (Compound 10)(R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid

In a 25 mL, 3-neck round-bottom flask, (R)-2-amino-3-methylbutanoic acid(0.8 g, 1.0 eq.) was dissolved in water (4 mL). Sodium bicarbonate(0.394 g, 1.1 eq.), followed by di-tert-butyl dicarbonate (1.86 g, 2.0eq.) was added and the reaction mixture was stirred for 2 h at RT. Thereaction mixture was extracted with EtOAc (3×10 mL). The combinedorganic layers were washed with aqueous saturated sodium chloridesolution (25 mL), dried over anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure (25° C., 20 mmHg) to afford 0.75 gof the crude product that was purified by column chromatography (60/120silica gel) using methanol/dichloromethane gradient (the column waspacked in dichloromethane and the desired compound started eluting from3% methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (0.44 g, yield:47.3%).

(R,Z)-2-amino-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-3-methylbutanehydrazide2,2,2-trifluoroacetate (Compound 10)

In a 10 mL round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylohydrazide(0.05 g, 1.0 eq.) was dissolved in THF (5 mL) and cooled to −60° C.where (R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (0.038 g,1.3 eq.) was introduced dropwise. T3P (50% in EtOAc) (0.16 mL, 2 eq.)was added dropwise followed by DIPEA (0.095 mL, 4 eq.) and the reactionmixture was stirred for 1 h at −60° C. The reaction mixture wasconcentrated under reduced pressure (25° C., 20 mm Hg) to afford thecrude product that was purified by column chromatography (60/120 silicagel) using methanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford (R,Z)-tert-butyl(1-(2-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)hydrazinyl)-3-methyl-1-oxobutan-2-yl)carbamate(0.017 g, yield: 26%). In a 10 mL round-bottom flask, (R,Z)-tert-butyl(1-(2-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)hydrazinyl)-3-methyl-1-oxobutan-2-yl)carbamatewas then dissolved in dichloromethane (2 mL). TFA (0.2 mL) was added andthe reaction mixture was stirred at RT for 5 h. The reaction mixture wasconcentrated under reduced pressure (25° C., 20 mm Hg) to afford thecrude product (0.02 g), which was triturated with petroleum ether anddried under reduced pressure to yield(R,Z)-2-amino-N′-(3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acryloyl)-3-methylbutanehydrazide2,2,2-trifluoroacetate (0.007 g, yield: 35%).

Example 11. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(pyrazin-2-yl)acetyl)acrylohydrazide(Compound 11) 2-(pyrazin-2-yl)acetohydrazide

In a 25 mL sealed tube, methyl 2-(pyrazin-2-yl)acetate (0.25 g, 1.0 eq.)was dissolved in ethanol (5 mL) at RT. Hydrazine hydrate (0.33 g, 4 eq.)was introduced dropwise at RT and the reaction mixture was heated at120° C. for 20 h. The reaction mixture was concentrated under reducedpressure (40° C., 20 mm Hg) to afford the crude2-(pyrazin-2-yl)acetohydrazide (0.2 g) which was used without furtherpurification in the following step.

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(pyrazin-2-yl)acetyl)acrylohydrazide(Compound 11)

In a 50 mL, 3-neck round-bottom flask,(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.3 g, 1.0 eq.) was dissolved in EtOAc:THF (15 mL; 2:1) and cooledto −60° C. where 2-(pyrazin-2-yl)acetohydrazide (0.129 g, 1.1 eq.) wasintroduced. T3P (50% in EtOAc) (1.01 mL, 2 eq.) was added dropwisefollowed by DIPEA (0.35 mL, 2.5 eq.) and the reaction mixture wasstirred for 1 h at −60° C. The reaction mixture was concentrated underreduced pressure (25° C., 20 mm Hg) to afford the crude product that waspurified by column chromatography (60/120 silica gel) usingmethanol/dichloromethane gradient (the column was packed indichloromethane and the desired compound started eluting from 3%methanol/dichloromethane). Fractions containing the desired compoundswere combined to afford(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(pyrazin-2-yl)acetyl)acrylohydrazide(0.025 g, yield: 5%).

Example 12. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-morpholino-2-oxoacetyl)acrylohydrazide(Compound 12)

Synthesis of ethyl 2-morpholino-2-oxoacetate

A solution of ethyl 2-chloro-2-oxoacetate (1.25 g, 9.18 mmol) in diethylether (5 mL) was added dropwise to a solution of morpholine (1.0 g,11.48 mmol) in diethyl ether (20 mL) and triethylamine (1.16 g, 11.48mmol) at 0° C. The reaction mixture was allowed to warm to roomtemperature and stirred for 2 h. The reaction mixture was filtered andthe filtrate was concentrated under reduced pressure. The yellow-coloredoil was transferred into 25 mL iced water and extracted with ethylacetate (3×20 mL). Combined organic layers were washed with brine, driedover anhydrous sodium sulphate, and concentrated under reduced pressureto give 1 g of the crude product, which was used further without anypurification. Crude yield 47%. ¹H NMR (400 MHz, CDCl₃) δ 4.33-4.38 (q,2H), 3.72-3.76 (m, 4H), 3.65-3.68 (m, 2H), 3.47-3.50 (m, 2H), 1.37-1.40(t, 3H). LCMS m/z 187.93 [M+H]⁺, t_(R)=0.525 min.

Synthesis of 2-morpholino-2-oxoacetohydrazide

Ethyl 2-morpholino-2-oxoacetate (1.0 g, 5.34 mmol) was dissolved inethanol (7 mL) and hydrazine hydrate (0.267 g, 5.34 mmol) was addeddropwise at 0° C. The reaction mixture was stirred at room temperaturefor 1.5 h. The reaction mixture was concentrated under reduced pressureto give 0.9 g of the crude product, which was used without furtherpurification in the following step. Crude yield 90%. ¹H NMR (400 MHz,CDCl₃) δ 9.79 (s, 1H), 4.43-4.48 (m, 2H), 3.56-3.61 (m, 4H), 3.40-3.48(m, 4H). LCMS m/z 174.16 [M+H]⁺, t_(R)=2.031 min.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-morpholino-2-oxoacetyl)acrylohydrazide

A solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.2 g, 0.569 mmol) and 2-morpholino-2-oxoacetohydrazide (0.02 g,0.175 mmol) in THF (3 mL) was cooled to −60° C. T₃P (0.098 g, 0.569mmol) (0.50 mL) was added dropwise followed by DIPEA (0.11 g, 0.854mmol) and stirred at −60° C. for 1 h. The reaction mixture wastransferred into 25 mL of iced water and extracted with ethyl acetate(2×25 mL). Combined organic layers were washed with brine, dried overanhydrous sodium sulphate, and concentrated under reduced pressure togive 0.3 g of crude product, which was purified by chromatography (0-4%MeOH/CH₂Cl₂) to give 0.15 g of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-morpholino-2-oxoacetyl)acrylohydrazide (Yield 50%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.70-10.88 (m,2H), 9.56 (s, 1H), 8.57 (s, 2H), 8.29 (s, 1H), 7.52-7.55 (d, J=10.4 Hz,1H), 6.0-6.03 (d, J=10.4 Hz, 1H), 3.51-3.64 (m, 8H). LCMS m/z 507.25[M+H]⁺, t_(R)=2.012 min.

Example 13. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3,5-dimethylmorpholino)acetyl)acrylohydrazide(Compound 13)

Synthesis of 2,2′-azanediyldipropan-1-ol

2-Aminopropan-1-ol (5 g, 66.57 mmol) and 1-hydroxypropan-2-one (5.77 g,77.89 mmol) were dissolved in ethanol (115 mL) and 50 mg of PtO₂ wasadded. The reaction mixture was stirred at 50 psi H₂ pressure at roomtemperature for 24 h. The reaction mixture was filtered and the filtratewas concentrated under reduced pressure to give the crude product, whichwas used without further purification in the following step. Crudeyield: 79%. ¹H NMR (400 MHz, CDCl₃) δ 4.45 (bs, 2H), 3.42-3.43 (m, 1H),3.16-3.22 (m, 4H), 2.65-2.69 (m, 2H) 0.87-0.91 (m, 6H): LCMS m/z 133.99[M+H]⁺, t_(R): 4.077 min.

Synthesis of 3,5-dimethylmorpholine

2,2′-Azanediyldipropan-1-ol (7 g, 52 mmol) was suspended in Conc. H₂SO₄(5.3 mL, 99.8 mmol) at room temperature and heated at 180° C. for 8 h.The reaction mixture was cooled at 0° C. and solution of KOH (11.79 g,21.02 mmol) in 60 mL water was added dropwise. The reaction mixture wasstirred at room temperature for 12 h. The reaction mixture was filteredand filtrate was extracted with CHCl₃:MeOH (85:15; 5×50 mL). Thecombined organic layers were dried over anhydrous sodium sulphate andconcentrated under reduced pressure to give 3.5 g of crude product,which was used without further purification in the following step (CrudeYield: 58%).

Synthesis of ethyl 2-(3,5-dimethylmorpholino)acetate

Potassium carbonate (0.311 g, 2.25 mmol) and ethyl bromoacetate (0.319g, 1.91 mmol) was added to the solution of 3,5-dimethylmorpholine (0.2g, 1.73 mmol) in acetonitrile (4 mL) at room temperature. The reactionmixture was stirred at 60° C. for 12 h. The reaction mixture wastransferred into iced-water and extracted with ethyl acetate (20 mL×3).The combined organic layers was washed with brine, dried over anhydrousNa₂SO₄, and concentrated under reduced pressure to give the crudeproduct, which was used in the next step without further purification(Crude Yield: 54%).

Synthesis of ethyl 2-(3,5-dimethylmorpholino)acetohydrazide

Ethyl-2-(3,5-dimethylmorpholino)acetate (0.19 g, 0.944 mmol) wasdissolved in ethanol (4 mL) and hydrazine hydrate (0.047 g, 0.944 mmol)was added dropwise. The reaction mixture was stirred at 80° C. for 20 hand the reaction mixture was concentrated under reduced pressure to givethe crude product, which was used without further purification in thesubsequent step. (Crude yield: 97%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.95(s, 2H), 8.84 (s, 1H), 3.60-3.63 (m, 2H), 3.25-3.29 (m, 2H), 3.14 (s,2H), 3.05 (s, 2H), 0.86-0.88 (m, 6H): LCMS m/z 188.12 [M+H]⁺, t_(R)4.716 min.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3,5-dimethylmorpholino)acetyl)acrylohydrazide

To the solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.2 g, 0.569 mmol) and 2-(3,5-dimethylmorpholino)acetohydrazide(0.106 g, 0.569 mmol) in THF (10 mL) were added T3P (0.543 g, 0.854mmol) followed by DIPEA (0.110 g, 0.854 mmol) at −60° C. and stirred for2 h. The reaction mixture was transferred into 25 mL iced-water andextracted with ethyl acetate (2×25 mL) and the combined organic layerswas washed with brine, dried over anhydrous sodium sulphate, andconcentrated under reduced pressure to afford the crude product, whichwas purified by chromatography (0-3% MeOH/CH₂Cl₂) to give 0.02 g of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3,5-dimethylmorpholino)acetyl)acrylohydrazide(Yield: 7%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.58 (s, 1H), 9.83 (s, 1H),9.56 (s, 1H), 8.54-8.56 (m, 2H), 8.25-8.30 (m, 1H), 7.49-7.51 (d, J=10.4Hz, 1H)), 6.01-6.04 (d, J=10.4 Hz, 1H), 3.44-3.57 (m, 2H), 3.28-3.34 (m,2H), 3.21 (s, 1H), 3.15 (s, 1H), 2.84-2.88 (m, 2H), 0.93-1.04 (m, 6H):LCMS m/z 521.18 [M+H]⁺, t_(R) 1.898 min.

Example 14. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3-oxomorpholino)acetyl)acrylohydrazide(Compound 14)

Synthesis of Ethyl 2-(3-oxomorpholino) acetate

Morpholin-3-one (3 g, 29.67 mmol) was dissolved in DMF (15 mL, 29.67mmol) and NaH (1.78 g, 44.51 mmol) was added at 0° C. The reactionmixture was stirred at room temperature for 30 min and ethylbromoacetate (3.76 mL, 32.64 mmol) was added dropwise. The reaction mixturewas further stirred at room temperature for 3 h and transferred into 50mL water and extracted with EtOAc (3×50 mL). The combined organic layerswas washed with brine solution (2×50 mL), dried over anhydrous sodiumsulphate and concentrated under reduced pressure to give the crudeproduct, which was purified by chromatography (0-100% ethylacetate/hexane) to give 600 mg of ethyl-2-(3-oxomorpholino)acetate(Yield: 10%). LCMS m/z 187 [M+H]⁺, t_(R)2.505 min.

Synthesis of 2-(3-oxomorpholino)acetohydrazide

Ethyl-2-(3-oxomorpholino)acetate (600 mg, 3.21 mmol) was dissolved inethanol (3 mL) and hydrazine hydrate (160.46 mg, 3.21 mmol) was added atroom temperature. The reaction mixture was heated at 80° C. for 1 h. Thereaction mixture was transferred into 50 mL water and extracted withEtOAc (3×50 mL). The combined organic layers was washed with brine,dried over anhydrous sodium sulphate, and concentrated under reducedpressure to give the crude product, which was used without furtherpurification in the subsequent step (Crude yield: 54%). LCMS m/z 174.05[M+H]⁺ t_(R) 2.489 min.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3-oxomorpholino)acetyl)acrylohydrazide

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.400 g, 1.14 mmol) was dissolved in THF(4 mL) and2-(3-oxomorpholino)acetohydrazide (0.295 g, 1.71 mmol) was added. T₃P(1.09 g, 1.71 mmol) was added dropwise followed by DIPEA (220.80 mg,1.71 mmol) at −60° C. and the reaction mixture was stirred for 1 h. Thereaction mixture was transferred into 25 mL iced-water and extractedwith EtOAc (2×25 mL). Combined organic layers was washed with brine,dried over anhydrous sodium sulphate, and concentrated under reducedpressure to give the crude product which was purified by chromatography(0-4% MeOH/CH₂Cl₂) to give 0.05 g of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3-oxomorpholino)acetyl)acrylohydrazide (Yield: 8%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.33 (bs, 2H),9.63 (s, 1H), 8.57 (s, 2H), 8.30 (s, 1H), 7.50-7.52 (d, J=8 Hz, 1H)),6.01-6.03 (d, J=8 Hz, 1H), 4.08-4.12 (m, 4H), 3.85-3.87 (m, 2H),3.41-3.44 (m, 2H). LCMS m/z 507.13 [M+H]⁺, t_(R) 1.950 min.

Example 15. Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3,3-dimethylmorpholino)acetyl)acrylohydrazide(Compound 15)

Synthesis of Ethyl 2-(3,3-dimethylmorpholino)acetate

3,3-Dimethylmorpholin (1 g, 8.68 mmol) was dissolved in acetonitrile (5mL) and potassium carbonate (1.8 g, 13 mmol) was added. The reactionmixture was stirred at room temperature for 30 min and ethylbromoacetate (1.1 mL, 9.55 mmol) was added. The reaction mixture was heatedat 60° C. for 1 h. Then reaction mixture was transferred into 50 mLwater and extracted with ethyl acetate (3×50 mL). The combined organiclayers was washed with brine, dried over anhydrous sodium sulphate, andconcentrated under reduced pressure to give the crude product, which wasused without further purification in the next step (Crude yield: 91%).LCMS m/z 202.9 [M+H]⁺, t_(R) 2.33 min.

Synthesis of 2-(3,3-dimethylmorpholino)acetohydrazide

To the solution of ethyl 2-(3-oxomorpholino)acetate (600 mg, 2.98 mmol)in ethanol (3 mL) hydrazine hydrate (0.20 mL, 2.98 mmol) was added atroom temperature. The reaction mixture was heated at 80° C. for 1 h,allowed to cool to room temperature, transferred into 50 mL water, andextracted with ethyl acetate (3×25 mL). The combined organic layers waswashed with brine, dried over anhydrous sodium sulphate, andconcentrated under reduced pressure to give the crude product, which wasused without further purification in the following step (Crude yield:28%). LCMS m/z 188 [M+H]⁺ t_(R): 188 min.

Synthesis of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3,3-dimethylmorpholino)acetyl)acrylohydrazide

To the solution of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylicacid (0.250 g, 0.7 mmol) and 2-(3,3-dimethylmorpholino)acetohydrazide(0.160 g, 0.85 mmol) in THF (2.5 mL) T₃P (0.63 mL, 1.06 mmol) was addeddropwise followed by DIPEA (0.18 mL, 1.06 mmol) at −60° C. The reactionmixture was stirred for 1 h, transferred into 25 mL iced-water, andextracted with ethyl acetate (2×25 mL). The combined organic layers waswashed with brine, dried over anhydrous sodium sulphate, andconcentrated under reduced pressure to give the crude product, which waspurified by chromatography (0-4% MeOH:CH₂Cl₂) to give 0.05 g of(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N′-(2-(3,3-dimethylmorpholino)acetyl)acrylohydrazide (Yield: 13%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.55(s, 1H), 9.81 (s, 1H), 9.62 (s, 1H), 8.56 (s, 2H), 8.29 (s, 1H),7.49-7.51 (d, J=10.4 Hz, 1H)), 6.01-6.03 (d, J=10.4 Hz, 1H), 3.65-3.67(m, 2H), 3.30-3.34 (m, 2H), 3.08 (bs, 2H), 2.55-2.58 (m, 2H), 0.96 (s,6H). LCMS m/z 521.18 [M+H]⁺, t_(R) 1.937 min.

Example 16. Assays

Certain compounds of the invention, along with Compounds X-1, X-2 andX-3 (shown below) were tested in various assays.

Inhibition of Nuclear Export

The inhibition of CRM1 mediated nuclear export by compounds of theinvention was determined. The results are shown in Table 2. Theinhibitory activity of compounds for the CRM1 protein was determined inthe RevGFP assay. Compounds of the invention are active in Rev-GFP assaywith IC₅₀<10 μM with the most preferred compounds having activities withIC₅₀ values of 1 μM.

Experimental protocol: Rev is a protein from human immunodeficiencyvirus type 1 (HIV-1) and contains a nuclear export signal (NES) in itsC-terminal domain and a nuclear localization signal (NLS) in itsN-terminal domain. Nuclear export of Rev protein is dependent on theclassical NES/CRM1 pathway (Neville et al, 1997, Kau et al, 2003).Nuclear accumulation of Rev is observed in cells treated with specificinhibitors of CRM1, such as LMB (Kau et al, 2003). In this assay,U2OS-RevGFP cells are seeded onto clear-bottom, black, 384-well platesthe day before the experiment. Compounds are serially diluted 1:2starting from 40 M in a separate 384-well plate in DMEM, and thentransferred onto cells. Cells are incubated with compound for ˜1 hrbefore fixation with 3.7% formaldehyde and nuclei staining with Hoechst33258. The amount of GFP in cell nuclei was measured and compound IC₅₀swere determined (Kau et al, 2003).

MTT Cell Proliferation Assay

The CellTiter 96® AQueous One Solution cell proliferation assay(Promega) was used on MM1.S, Jurkat and HCT-116 cells to study thecytotoxic and cytostatic properties of the compounds. The assay is basedon the cleavage of the tetrazolium salt, MTS, in the presence of anelectron-coupling reagent PES (phenazine ethosulfate). The MTStetrazolium compound is bioreduced by cells into a colored formazanproduct that is soluble in tissue culture medium. This conversion ispresumably accomplished by NADPH or NADH produced by dehydrogenaseenzymes in metabolically active cells. Assays are performed by adding asmall amount of the CellTiter 96® AQueous One solution reagent directlyto culture wells, incubating for 1-4 hours and then recording theabsorbance at 490 nm with a 96-well plate reader. The absorbancerevealed directly correlates to the cell number and their metabolicactivity. The cells were seeded at 5×10³ to 1.5×10⁴ cells (depending oncell type) in each well of 96-well plate in 100 μL of fresh culturemedium and adherent cells were allowed to attach for overnight. Thestock solutions of the compounds were diluted in cell culture medium toobtain eight concentrations of each drug, ranging from 1 nM to 30 μM andDMSO at less than 1% v/v was used as a negative control. After 72 h oftreatment 20 μl of CellTiter 96® AQueous reagent was added into eachwell of the 96-well assay plates and the plate was incubate at 37° C.for 1-4 hours in a humidified, 5% CO2 atmosphere. Then the absorbance ofeach well was recorded at 490 nm by using a 96-well plate reader. Inmost cases the assay was performed in triplicates and the results werepresented as half maximal inhibitory concentration (IC₅₀) describedbelow. Optical density versus compound concentration was plotted andanalyzed using non linear regression equations (Excel Fit) and the IC₅₀for each compound was calculated. The results are shown in Table 2.

Determination of Pharmacokinetics (PK) and Brain:

Plasma Ratio Blood was collected from mice (N=3) to contribute to thetotal of 10 time points (pre-dose, 5 min, 15 min, 30 min, 1 hour, 2hours, 4 hours, 8 hours, 12 hours and 24 hours post dose). Mice werebled on a rotating basis, each mouse contributing 3 time points to theblood collection. At the designated time points, animals wereanaesthetized under isoflurane, and approximately 110 μL of blood pertime point was collected via retro-orbital puncture into pre-cooledK₂EDTA (anti-coagulant) tubes. Blood samples were put on wet ice andcentrifuged (2000 g, 5 min at 4° C.) to obtain plasma within 30 minutesof sample collection. All samples were stored frozen at approximately−80° C. until analysis. Prior to analysis, samples were mixed withinternal standard (dexamethasone) in acetonitrile, vortexed,centrifuged, and supernatant was injected for analysis. Concentration ofcompounds in plasma was determined using LC-MS-MS instrumentation (API4000, Triple Quadruple with electrospray ionization; Acuity UltraPerformance Liquid Chromatography column C18, with MeOH and formic acidas organic solvents). PK parameters including but not limited to Tmax,Cmax, t_(1/2), AUC_(last), AUC_(inf) were calculated using WinNonlinProfessional 6.2 software package, non-compartmental pharmacokineticmodel NCA200.

Brain to Plasma Ratio (B:P).

A separate group of mice (N=3) were dosed (PO at 10 mg/kg unlessotherwise indicated) and then sacrificed at the time of maximal plasmaconcentration (estimated T_(max) at 2 hours post-dose) where terminalplasma and brain were collected. Brain tissue following collection wasrinsed with cold saline, dried on filter paper, weighed and snap-frozenby placing on dry ice. All samples were stored frozen at approximately−80° C. until analysis. At the time of analysis, brain tissue washomogenized (homogenizing solution PBS, pH 7.4), mixed with internalstandard (dexamethasone) in acetonitrile, vortexed, centrifuged, andsupernatant was injected for analysis of compound concentration usingLC-MS-MS methodology (API 4000, Triple Quadruple with electrosprayionization; Acuity Ultra Performance Liquid Chromatography column C18,with MeOH and formic acid as organic solvents). Plasma samples weretreated with the identical method (except homogenization step) andconcentration of compound in either matrix was calculated based on thegenerated standard curves. The results are shown in Table 2.

TABLE 2 Assay Results for Compounds of Formula I and ComparatorsThereto. Rev Export Cytotoxicity AUC_(Inf) Compound [IC₅₀] [IC₅₀] (hr ·ng/mL)* B:P* X-1** <1 μM <1 μM  209^(‡) NT X-2*** <1 μM <1 μM   68.3^(†)1.27^(†) X-3 <1 μM <1 μM 12300 5.0  1 NT <1 μM 33100 2.25  2 <1 μM <1 μM28900 0.16  3 <1 μM <1 μM 15200 0.03  4 NT <1 μM 20929 0.028  5 NT <1 μMNT NT  6 NT <1 μM NT NT  7 NT <1 μM NT NT  8 NT <1 μM  9150 0.41 19 NT<1 μM  671**** N/A 10 NT <1 μM NT NT 11 NT <1 μM  8340 0.095 12 <1 μM <1μM 19600 0.06 13 NT <1 μM  1103 1.5 14 NT <1 μM  1419 0 15 NT <1 μM  5880 *Dosed in mice at 10 mg/kg po. **Compound 26 from US 2009/0275607.***Compound 44 from US 2009/0275607. ****Dosed in mice at 5 mg/kg po^(‡)AUC_(Inf) values for compound X-1 dosed in mice at 10 mg/kg po werebelow limit of quantitation. Data reported for 5 mg/kg iv. ^(†)Dosed inrats at 10 mg/kg po. NT = not tested N/A= below quantifiable limit

The AUC_(Inf) for compound X-1 was below the limit of detection whendosed in mice at 10 mg/kg po. When dosed at 5 mg/kg iv, compound X-1showed minimal exposure, as indicated by the low AUC_(Inf) of 209hr·ng/mL. The brain to plasma ratio for compound X-1 was not determineddue to its negligible levels (below the quantitation limit) in the brainwhen dosed po.

The AUC_(Inf) for compound X-2 was calculated to be 68.3 hr·ng/mL whendosed in rats at 10 mg/kg po. Such exposure levels are exceedingly lowwhen compared to compound X-3 and compounds of Formula I of the presentinvention. However, compound X-2 exhibits a moderate brain to plasmaratio. The low AUC_(Inf) coupled with a non-negligible brain to plasmaratio suggests that compound X-2 can crosses the BBB despite the lowexposure levels. Applicants believe that Compound X-2 would have asignificantly higher brain to plasma ratio if its AUC_(Inf) wereincreased.

The AUC_(Inf) for compound X-3 was calculated to be 12300 hr·ng/mL whendosed in rats at 10 mg/kg po, indicated good exposure. However, X-3demonstrated a high B:P ratio of 5.0.

The compounds of Formula I, all show a high AUC_(Inf) (>3500 hr·ng/mL)and a relatively low B:P (<2.5). Generally, greater exposure levels of atherapeutic agent often increase the likelihood of brain penetration. Itis therefore surprising and unexpected that compounds of formula Iexhibit high AUC_(Inf) levels while relatively low brain to plasmaratios.

Example 17. Models Evaluation of the Effects of Compound 2 on TumorGrowth in the Z-138 Lymphoma Cell Line Grown as a Xenograft in SCID Mice

Z-138 (ATCC # CRL-3001) mantle cell lymphoma cells were obtained fromATCC. These cells were grown in IMEM medium supplemented with 10% horseserum, 1% penicillin and streptomycin, and 2 mM L-glutamine. Cells weresub-cultured by dilution at a ratio of 1:5 to 1:10. Twenty-four (24)female CB-17 SCID mice (Charles River Labs strain code 236), aged 5 to 6weeks were used. The SCID mice were inoculated in the left flank withZ-138 cells in a volume of 0.2 mL, equivalent to 4×10⁷ cells per mouse.

Treatment was initiated when the tumors reached a mean volume of 84.3mm³. Mice were allocated to four (4) groups of eight (8) prior to theinitiation of treatment based on tumor volume such that mean tumorvolume in each group was within the range of 77 to 92 mm³. Mice weretreated with vehicle, standard of care drug/positive control drug(cyclophosphamide) or Compound 2, as shown in Table 3.

TABLE 3 Initial Study Groups Number Route of of Adminis- Group animalsTest Article Dose tration Schedule 1 8 Vehicle 10 ml/kg PO MWF 2 8Cyclo- 80 mg/kg IP Days phosphamide 1, 3, 5 3 8 Compound 2 15 mg/kg POMWF 4 8 Compound 2 7.5 mg/kg PO MWF

Animals were fed with Labdiet® 5001 rodent chow and sterile water adlibitum. Tumors were measured once every two days with micro-calipers,and tumor volume was calculated as (length×width×width)/2. All animalswere weighed every day in order to assess possible differences in animalweight among treatment groups as an indication of possible toxicityresulting from the treatments. Animals with weight loss of more than 20%of their starting weight were euthanized. Mice with weight loss of morethan 15% of their starting weight were not treated again until weightloss recovered to less than 5% of their starting weight. Any animalswith a tumor volume of more than 1500 mm³ were euthanized.

Dosing solutions were prepared fresh on each day of dosing. Compound 2was supplied as a lyophilized powder containing 69.61% Compound 2 withthe balance made up of Pluronic F-68 and PVP K29/32. This was preparedby dissolving the lyophilized powder in sterile water. Cyclophosphamidewas dissolved at 8 mg/mL in sterile water for injection. All testarticles were administered in a volume of 10 mL/kg body weight.

Statistical differences between treatment groups were determined usingMann-Whitney Rank Sum or ANOVA tests with a critical value of 0.05.

FIG. 1 shows that all treatment groups showed statistically significantreductions in tumor growth relative to vehicle when evaluated bycomparing the area under the growth curves using an ANOVA test for bothtumor volume and percent tumor volume. These treatment groups showedsignificant tumor growth reductions at p<0.0001. Some weight loss wasobserved in the group treated with Compound 2 at 15 mg/kg and, althoughstatistically significant, when compared to vehicle controls, severeweight loss was limited to a few animals.

Compound 2, administered orally, had antitumor effect at both 7.5 mg/kgand 15 mg/kg doses in a dose dependent manner.

Anti-Tumor Activity of Compound 2 in the A549 Small Cell Lung CarcinomaModel

The A549 cell line was derived from explant culture of alveolarcarcinoma tissue from a 58-year-old Caucasian male. The cells were grownin Ham's F12-K tissue culture media with 10% fetal calf serum and 1%penicillin/streptomycin. Cells were routinely trypsinized and passaged1:10. Thirty-two (32) female CB-17 SCID mice (Charles River Labs straincode 236), aged 5 to 6 weeks were used with a mean pre-treatment bodyweight of 16.3 grams. Mice were divided into four (4) groups of eight(8) prior to the initiation of treatment based on tumor volume. On theday of implantation, cells were washed in PBS, trypsinized andresuspended in complete media to a density of 2×10⁷ cells/mL prior tobeing mixed with an equal volume of Matrigel. This mixture was theninoculated subcutaneously into mice in a volume of 0.1 mL using a 23Gneedle.

Mice were treated with vehicle, standard of care drug/positive controldrug (cisplatin) or Compound 2, as shown in Table 4. Animal weights andcondition were recorded daily, and tumors were measured on Mondays,Wednesdays and Fridays with micro-calipers, and tumor volume wascalculated as (length×width×width)/2.

TABLE 4 Initial Study Groups Number Route of of Admin- Group animalsTest Article Dose istration Schedule 1 8 Vehicle 10 ml/kg PO MWF 2 8Cisplatin  5 mg/kg IP Days 1, 15 3 8 Compound 2 10 mg/kg PO MWF 4 8Compound 2  5 mg/kg PO MWF

Animals with weight loss of more than 20% of their starting weight wereeuthanized. Mice with weight loss of more than 15% of their startingweight were not treated again until weight loss recovered to less than5% of their starting weight. Any animals with a tumor volume of morethan 1500 mm³ were euthanized.

Dosing solutions were prepared fresh on each day of dosing. Compound 2was supplied as a lyophilized powder containing 69.61% Compound 2 withthe balance made up of Pluronic F-68 and PVP K29/32. This was preparedby dissolving the lyophilized powder in sterile water. Cisplatin wasdissolved at 5 mg/mL in DMSO and diluted 1:10 in sterile water forinjection. All test articles were administered in a volume of 0.1 mL/10g body weight.

Statistical differences between treatment groups were determined usingMann-Whitney Rank Sum or ANOVA tests with a critical value of 0.05.

The data for tumor volume change during the study are shown in FIG. 2.The mean tumor volume for the vehicle control group increased from 95mm³ on Day 1 to 1669 mm³ on Day 29. The group treated with cisplatin hada mean tumor volume of 104 mm³ on Day 1, increasing to 1136 mm³ on Day29. Mice treated with Compound 2 at 10 mg/kg PO (Group 3) had a meantumor volume of 101 mm³ on Day 1, which increased to 686 mm³ by Day 29.Mice treated with Compound 2 at 5 mg/kg PO (Group 6) had a mean tumorvolume of 101 mm³ on Day 1, which increased to 1231 mm³ by Day 29.

Additional analyses of the tumor volume data were performed bycalculating the mean area under the curve (AUC) for each tumor andcomparing the groups using a one-way ANOVA test. This analysis indicatedthat there were statistically significant differences between thevehicle control group and the group treated with Compound 2 at 10 mg/kg(p=0.0005). It should be noted that there was that there was nostatistically significant reduction in tumor growth in the positivecontrol group (cisplatin).

Compound 2, administered orally, had an antitumor effect at both 5 mg/kgand 10 mg/kg doses in a dose dependent manner. However, it was only the10 mg/kg group that showed a statistically significant difference whencompared to the vehicle treated group.

Evaluation of Compound 2 in the Anti-Collagen Antibody Induced MouseModel of Rheumatorid Arthritis (CAIA)

Twenty-four (24) male Balb/c mice, aged 6 to 8 weeks were used. Theweight variation of animals at the time of treatment initiation did notexceed ±20% of the mean weight. Animals were randomly assigned to 3groups that would receive vehicle, dexamethasone or Compound 2. On studyDay 0 (study commencement), all mice were subjected to a 2 mgintravenous injection of ArthritoMAb™ antibody cocktail (MD Biosciences#S1203001) followed by an intraperitoneal injection of LPS (100 g/mouse)on study Day 3. Study animals were treated with 7.5 mg/kg Compound 2 or4 mg/kg Compound 2 orally; 1 mg/kg dexamethasone intraperitoneally; orvehicle orally. Treatments were administered once daily on days 4, 6, 8and 10 for all groups, except where dosing vacations applied. If ananimal's weight dropped below 87% of its day 0 starting weight, theanimal was not dosed until it gained weight equivalent to 90% or more ofday 0 weight.

Arthritis development, clinical signs and body weights were monitored inall mice on study days 0, 3-8, 10 and 12. Observations included changesin skin, fur, eyes, mucous membranes, occurrence of secretions andexcretions (e.g., diarrhea) and autonomic activity (e.g., lacrimation,salivation, piloerection, pupil size, unusual respiratory pattern). Allpaws (front left and right, and rear left and right) of each animal wereexamined for signs of arthritogenic responses prior to arthritisinduction and test item or control item administration on study Day 0and subsequently on study Days 3-8, 10 and 12 (study termination). Arthritis reactions were scored and recorded according to a 0-4 scale inascending order of severity, as shown Table 5 below. Paw thickness wasalso measured using a dial caliper (Kroeplin, Munich, Germany).

TABLE 5 Arthritis clinical score Arthritis Score Grade No reaction,normal 0 Mild, but definite redness and swelling of the ankle/wrist 1 orapparent redness and swelling limited to individual digits, regardlessof the number of affected digits Moderate to severe redness and swellingof the ankle/wrist 2 Redness and swelling of the entire paw includingdigits 3 Maximally inflamed limb with involvement of multiple joints 4

The dose administered was calculated based on the assumption that theanimals weighed, on average, 20 g. A stock solution of dexamethasone wasprepared in 100% ethanol and diluted to the appropriate concentration inPBS prior to use. Vehicle for the vehicle control group was prepared bydissolving 0.6 g Pluronic and 0.6 g PVP in 100 mL distilled deionisedwater. The MAb stock solution (10 mg/mL) was supplied by MD Biosciences,Division of Morwell Diagnostics GmbH. LPS was diluted with PBS toachieve the appropriate concentration. Thorough vortexing was requiredjust prior to its injection. Compound 2 was supplied as a lyophilizeddrug powder containing 70.71% Compound 2 with the balance made up ofPluronic F-68 and PVP K29/32. A fixed volume of 200 μL was administeredto each mouse.

Evaluation was primarily based on the mean values for arthritis scoringand paw thickness measurements. Where appropriate, analysis of the databy ANOVA with Tukey post hoc analysis was applied to determinesignificance of treatment effects.

FIGS. 3A and 3B show the results of the CAIA mouse model experiments.Clinical signs associated with LPS-administration developed in allgroups following the LPS boost on day 3. Compared to vehicle treatedmice, mice treated with 7.5 mg/kg or 4 mg/kg Compound 2 hadsignificantly reduced total arthritis scores on days 5-12 and 6-12,respectively. Dexamethasone treatment significantly reduced totalarthritis score compared to the vehicle group on days 6-12. Compared tovehicle treated mice, mice treated with 7.5 mg/kg or 4 mg/kg Compound 2had significantly reduced rear paw arthritis scores on days 5-12.Dexamethasone treatment significantly reduced rear paw arthritis scorecompared to the vehicle group on days 5 and 12. There were nosignificant differences in body weight between the vehicle-treated groupand test item-treated groups.

In view of the findings in the present study, Compound 2 at 7.5 mg/kg or4 mg/kg delivered orally exhibited significant anti-arthritic activityin the anti-collagen antibody induced model of rheumatoid arthritis,with sustained reductions in mean arthritis scores and reductions in pawthickness.

Efficacy Study of Compound 2 in Collagen-Induced Arthritis (CIA) inLewis Rats

Forty (40) female Lewis rats (BK), aged 6 to 8 weeks with apre-treatment body weight range of 180 to 200 g were divided randomlyinto four (4) groups (Groups A-D) of ten (10) rats each. The rats inGroups B to D were immunized intradermally with bovine CII in IFA atthree sites near the base of the tail and over the back with 500 μL ofthe emulsion on day 0 (200 μL, 200 μL, 100 μL for each site). On day 7,the rats in Groups B-D were given booster injections with the sameamount of the emulsion intradermally near the former injection sites. Inthe therapeutic treatment model (Groups C and D), dexamethasone orCompound 2 was orally administered to rats with CIA after the onset ofarthritis, as shown in Table 6. Rats were weighed daily and a drugholiday was given to an animal when a weight loss of greater than 13%.

TABLE 6 Initial study groups Group Immunization Treatment Administrationn A Naive PBS vehicle PO. QD, from 10 onset to day 28 B Model Col II invehicle PO. QD, from 10 IFA onset to day 28 C DEX Col II in DEX PO. QD,from 10 (1MPK) IFA (1MPK) onset to day 28 D Compound 2 Col II inCompound 2 PO. QoD, from 10 (4MPK) IFA (4MPK) onset to day 28

CIA development was evaluated via macroscopic scoring and measurementsof paw swelling. This was assessed every day for the first 5 days aftersensitization (day 7) and then twice per week (Monday and Thursday) forthe remaining time with the clinical scoring system for each paw shownin Table 7.

TABLE 7 Arthritis clinical scores Arthritis score Grade No evidence oferythema and swelling 0 Erythema and swelling confined to the mid-foot(tarsals) 1 or ankle joint Erythema and mild swelling extending from theankle to 2 the mid-foot Erythema and moderate swelling extending fromthe ankle 3 to the metatarsal Erythema and severe swelling encompass theankle, foot, 4 and digits

Foot volume was measured by plethysmometry on the same day of thearthritic measurement throughout the study period. The cubage of eachhind paw and swelling rate were measured and using the followingequation:

Swelling Rate=(C _(N) −C ₀)/C ₀×100%.

FIG. 4A is a graph of joint swelling versus time, and shows the jointswelling measured on a scale of 0-4 in naïve rats and rats treatedaccording to the model, with positive control, or with Compound 2.

Bovine CII (in 10 mM acetic acid) at 4 mg/mL was emulsified with anequal volume of IFA.

The clinical scores were summed for each animal, and the total averageof all animals in each group was expressed as the mean arthritic score.FIG. 4B is a graph of clinical scores as a function of time, and showsthe clinical arthritis scores of naïve rats and rats treated accordingto the model, with positive control, or with Compound 2.

On day 28 of the study, three representatives from each treatment groupwere euthanized and hind paws were harvested and stored in 4% neutralbuffered formalin. Prepared sections of hind paws were subjected tohematoxylin and eosin (H&E) staining.

Histopathological analysis of the control animals showed cartilageerosion and pannus formation in line with course of the disease.However, in the Compound 2 treated rats, relatively intact cartilage wasfound on the joint surface and pannus formation was minimal. The resultsof the histological analysis are shown in FIG. 5.

The Clinical Score, Joint Swelling and histological examination datashowed correlations. The results also showed the therapeutic efficacy ofCompound 2 at 4 mg/kg (MPK), as shown by its effects on Clinical Scores,Joint Swelling and histological examination. The results of the CIAmodel in Lewis rats are depicted in FIGS. 4A and 4B, and FIG. 5.

Anti-Psoriasis Activity of Compound 2 in phorbol-12-myristate-13-acetate(PMA)-Induced Psoriasis in Female BALB/C Mice

Twenty-four (24) female BALB/c mice, aged 6 to 8 weeks with a bodyweight between 22 and 30 g were used. The mice were randomized into four(4) groups of eight (8) mice each. The grouping of animals was asfollows: Group I (Naïve; ethanol), Group II (PMA; ethanol), Group III(PMA; Compound 2 10 μM) and Group IV (PMA; betamethasone). Twenty (20)μLof PMA (4 μg/20 μL of acetone) was applied topically on the uppersurface of pinna of ear of all animals in Group II to Group IV. PMA wasapplied daily on left ear and on alternate days (M-W-F) on right earfrom Day 1 to Day 9. Thirty (30) minutes after application of PMA,vehicle or standard compound (betamethasone) or Compound 2 was appliedtopically to the ears of animals from different groups. Of note,vehicle, standard compound, and Compound 2 were applied daily to bothears of different animals from Day 1 to Day 12.

Animals were observed daily for a period of 12 days for any treatmentrelated symptoms. Basal ear thickness was recorded in all animals(before application of PMA) using digital screw gauge at time TO (Day1). For the entire duration of the study, 4 hours after application ofvehicle, standard compound, or Compound 2, the thickness of the ears wasmeasured daily using digital screw gauge and scores of erythema, scalingand folding were recorded. Severity of damage to the pinna of ear wasassessed by the scoring systems shown in Table 8.

TABLE 8 Psoriasis scores Score Parameter 0 1 2 3 Erythema Normal MildModerate Severe Scaling Folding

The animals were supplied with nutritionally balanced autoclavedpelleted feed (Nutrivet Life Sciences, Pune (India)) ad libitum and hadaccess to normal drinking water throughout the experimental periods.

Commercially available 100% DMSO (LR Grade) and ethanol (LR Grade) wereused to prepare the formulations. PMA was prepared by dissolving 10 mgof PMA in 50.0 mL of acetone. Compound 2 was prepared by dissolving 1.47mg of Compound 2 in 300 μL of 100% DMSO.

The experimental results are expressed in FIGS. 6A-6D as mean±SEM. Therewas no significant difference between all treatment groups in bodyweight, food and water consumption. PMA application showed, (i) thethickness of left as well as right ear increased (Group II vs. Naïve)and (ii) the disease activity index (DAI) of left as well as right earincreased (Group II vs. Naïve). Importantly, topical application ofCompound 2 led to a prominent reduction in PMA-induced increases in (i)left and right ear thickness, and (ii) left and right ear DAI. Thiseffect was prominent on Days 6-8 of the study when more animals treatedwith Compound 2 had reduced left/right ear thickness (compared toanimals from Group II), and DAI (compared to animals from Group II). Ofnote, Compound 2-mediated reduction in PMA-induced increases inleft/right ear thickness and DAI diminished as the study progressed (Day10 and beyond).

In a PMA induced psoriasis model in mice, Compound 2 displayedstatistically significant anti-psoriatic activity.

Anti-Psoriasis Activity of Compound 2 in the Imiquimod (IMQ)-InducedDermal Inflammation/Psoriasis Model (STUDY 1)

Forty (40) male BALB/c mice aged 6 to 8 weeks were used with apretreatment body weight of 22 to 30 g. The BALB/c mice were randomizedinto four (4) groups of 10 mice per group. A small area (about 2×2 cm²)of skin on the dorsum of all the animals was neatly shaved. Group-Ianimals served as Naïve animals. Psoriasis was induced in Groups II toIV [Group II (IMQ; vehicle), Group III (IMQ; Compound 2 (1 μM)) andGroup IV (IMQ; cyclophosphamide (10 mg/kg)] by topical application of31.25 mg of IMQ cream daily on the dorsum of the animals from Day 1 toDay 13. Four hours after application of IMQ, vehicle or standardcompound (cyclophosphamide) or Compound 2 was administered (topically—30μL; orally—according to body weight) to the appropriate group from Day 1to Day 13 daily. Two hours after administration of vehicle or standardcompound or Compound 2, erythema, scaling, folding and thickening ofskin were recorded to determine the disease activity index (DAI).

Animals were observed daily for a period of 13 days for anytreatment-related symptoms. The daily observations included body weight,feed intake, skin thickening, scaling, folding, erythema, nasaldischarge, movement, respiration, hair, distended abdomen, skincondition, fur, mucous membrane, presence or absence of secretions, eyecondition, tail elevation, motor activity, posture and gait. Severity ofdamage to the dorsal portion of the skin was assessed by assigningerythema, scaling, folding and skin thickening scores based on externalobservations of skin, according to rubric in Table 9.

TABLE 9 Psoriasis scores Score Parameter 0 1 2 3 Erythema Normal MildModerate Severe Scaling Folding Skin thickening

Commercially available 100% DMSO (LR Grade), ethanol (LR Grade),cyclophosphamide (CMC), PVP and Pluronic were used to prepare theformulations. Compound 2 was prepared by dissolving 1.47 mg of Compound2 in 300 μL of 100% DMSO. Cyclophosphamide was prepared by dissolving500 mg of CMC in 100 mL distilled water.

The experimental results shown in FIGS. 7A and 7B are expressed asmean±SEM.

There was no significant difference in the body weight, food consumptionand water intake in the treatment group when compared to the controlgroup during the duration of the study. Compound 2 diminishedIMQ-induced disease manifestation.

Compound 2 shows anti-psoriatic activity, as evidenced by the reductionin disease activity index in comparison to the vehicle treated group.Further, Compound 2 caused this effect without adversely affecting bodyweight, food and water intake.

Anti-Psoriasis Activity of Compound 2 in the Imiquimod (IMQ)-InducedDermal Inflammation/Psoriasis Model (STUDY 2)

Forty (40) male BALB/c mice (Biological E Limited, Hyderabad (CPCSEAregistration number: 36/99/CPCSEA)) were divided into four (4) groupsconsisting of ten (10) mice each. Animals were randomized based on theirbody weight. The groups were designated as Group-I (Naïve), Group-II(IMQ; vehicle (PEG 400 and HPBCD)), Group-III (IMQ; Compound 2 (2.5mg/kg)) and Group-IX (IMQ; cyclophosphamide (10 mg/kg)).

A small area on the dorsum of each mouse was shaved, ensuring that theseareas were of equal size/area. Psoriasis was induced in Groups II to IVby topical application of 50 mg of IMQ cream daily from Day 1 to Day 6on the dorsum of the animals. On Day 1 and Day 2 of the study, fourhours after topical application of IMQ, Compound 2 or positive control(cyclophosphamide) or vehicle were administered to animals in pertinentgroups. Of note, animals in Group II and Group III were subjected tosubcutaneous injections, whereas animals in Group IV received oraladministration. The Compound 2, vehicle and cyclophosphamide treatmentwas terminated on Day 2. These groups of animals were maintained ondaily IMQ treatments until Day 6. On Day 7, the psoriasis-inducedanimals were re-randomized into 3 groups consisting of 10 animals eachbased on Cumulative Disease Activity Index (CDAI). From Day 7 to Day 9,animals received vehicle or positive control or Compound 2. Of note, onthese days animals were not treated with IMQ. From Day 10 to Day 14, theanimals were treated alternatively with IMQ (Days 10, 12 and 14), orvehicle, positive control or Compound 2 (Days 11, 13).

All animals were observed daily for a period of 16 days for grossobservations, body weight and feed and water intake. On Days 1 and 2,scorings of erythema, scaling, folding and thickening of skin wererecorded 2 hours after administration of vehicle/positive control/testcompounds, and on Days 3 to 14 scorings were recorded 4 hours after IMQapplication, or administration of positive control, vehicle or Compound2. The severity of induction on the dorsum of animal was assessed andscored as shown in Table 10.

Vehicle was prepared by dissolving 40 mg of HPBCD in 70.0 mL ofdistilled water. Compound 2 was prepared by dissolving 3.59 mg in 0.5%PVP and 0.5% Pluronic. Cyclophosphamide was prepared by dissolving 500mg of CMC in 100 mL distilled water.

The experimental results shown in Table 10 are expressed as mean±SEM.Data was assessed using one-way ANOVA, and post hoc analysis wasperformed using Dunnett's test.

TABLE 10 Rate of reduction of Disease Activity Index (DAI) Rate ofreduction of DAI Pre-IMQ/drug administration readings % change from 1stdose administration (i.e., from pre-IMQ/drug administration readings onDay 7) Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15Naïve, Vehicle 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 IMQ, Vehicle 0.0 4.2−33.3 −66.7 −60.4 −54.2 −45.8 −43.8 −43.8 IMQ, Compound 2 0.0 −33.8−58.8 −88.2 −73.5 −80.9 −61.8 −66.2 −64.7 IMQ, cyclophosphamide 0.0 16.3−39.5 −79.1 −25.6 −39.5 −25.6 −34.9 −41.9 4 hr. post-IMQ/drugadministration readings % change from 1st dose administration (i.e.,from pre-IMQ/drug administration readings on Day 7) Day 7 Day 8 Day 9Day 10 Day 11 Day 12 Day 13 Day 14 Naïve, Vehicle 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 IMQ, Vehicle 4.2 8.3 −35.4 −70.8 −58.3 −52.1 −39.6 −41.7IMQ, Compound 2 −30.9 −35.3 −63.2 −86.2 −70.6 −73.5 −60.3 −63.2 IMQ,cyclophosphamide 23.3 20.9 −41.9 −86.0 −25.6 −34.9 −32.6 −32.6

It was observed that the rate of reduction of disease activity index inanimals treated with Compound 2 was significantly greater than thatobserved in animals treated with vehicle. There was no significantdifference in the body weight, food consumption and water intake in thetreatment group when compared to the control group during the durationof the study.

The results obtained indicate that treatment with Compound 2 diminishedIMQ-induced disease manifestation without greatly impacting theconsumption of food or water, and thereby not showing any effect on thebody weight of animals in the treated groups.

The Effect of Compound 2 in Zucker Rats

Twenty-one (21) male Zucker rats aged 7 months were allocated into 3groups of N=7 based on equivalent body weights and food intakes. Anadditional group of N=7 age matched Zucker lean controls were includedas a control. Body weights and food and water intakes were measured atapproximately the same time each day (14:30-15:30 h). On treatment days,dosing was at 14:30-15:30 h (approximately 2 hours before lights off).

The Zucker obese and lean controls were orally treated with vehicle (10mL/kg dose volume; 0.5% Pluronic F68 and 0.5% PVP K29/32 in water) oneach weekday. Both Compound 2 (1.5 mg/kg and 3 mg/kg) groups were orallytreated on each weekday (10 mL/kg dose volume; 0.5% Pluronic F68 and0.5% PVP K29/32 in water). Prior to the treatment phase, 4 days baselinedata were collected. The treatment phase was for 16 days and a washoutphase of 6 days was also included.

FIGS. 8A and 8B and FIG. 9 show the effects of Compound 2 on Zuckerrats. At baseline, there was no significant difference in body weightand daily food intake between the 3 Zucker obese groups. However, allgroups were significantly different from the Zucker lean group.

Compound 2 (1.5-3 mg/kg oral) produced a dose-related decrease in dailyfood intake and body weight over the 16 day treatment period compared tothe Zucker control group. Compound 2 treatment also significantlyincreased water intake measured over the same period. There was asignificant difference in body weight gain between the 3 mg/kg Compound2 group and the Zucker vehicle group. There was no significantdifference in weight gain between the 1.5 mg/kg Compound 2-treated groupand the Zucker vehicle group.

Compound 2 showed a dose dependent decrease in daily food effect withthe higher dose (3 mg/kg) being more effective than the 1.5 mg/kg dose.Further, the Compound 2 group at 3 mg/kg showed lower weight gain incomparison to the Zucker control group.

Effect of Compound 2 in Diet-Induced Obesity Model

Male Sprague-Dawley rats of age 2 months were placed on a high fat diet(Research Diets Inc., product code D12492, 60% kcal % fat) for 3 months.A group of age-matched rats were fed normal lab chow (LabDiet 5001, ˜13%kcal % fat), these animals served as controls for DIO group.

At age 4 months, and 2 months into placement of high fat diet, all ratswere allocated into 3 groups of N=7 based on equivalent body weights andfood intakes. Body weights and food and water intakes were measured atapproximately the same time each day. On treatment days, dosing was atapproximately 2 hours before lights off.

The DIO control group was treated with vehicle (oral, 10 mL/kg dosevolume; 0.5% Pluronic F68 and 0.5% PVP K29/32 in water) on each weekday.The Compound 2 1.5 mg/kg group was orally treated on each weekdaythroughout the treatment phase (dose 10 mL/kg dose volume; 0.5% PluronicF68 and 0.5% PVP K29/32 in water). The Compound 2 3 mg/kg group (10mL/kg dose volume; 0.5% Pluronic F68 and 0.5% PVP K29/32 in water) wasorally treated initially once daily on each weekday for week 1, thentwice weekly (Monday, Wednesday) for week 2. During treatment weeks 3and 4, Compound 2 3 mg/kg treatment continued twice weekly, exceptdosing was on Monday and Thursday.

Prior to the treatment phase, 3 days baseline data were collected. Thetreatment phase was for 4 weeks. A washout phase of 10 days was alsoincluded.

Compound 2 was supplied in powder form. The test compound had an activepercentage of 65.89%. Active percentage was adjusted using BEW of 1.437and prepared by dissolving into 0.5% w/v Pluronic F-68 and 0.5% w/v PVPK-29-32 vehicle solution. The vehicle solution was prepared on a weeklybasis while Compound 2 was prepared fresh every 2 days and stored at +4°C. Animals were dosed at a volume of 10 mL/kg. Individual doses werecalculated based on the most recent body weights to provide the propermg/kg/day dosage.

FIGS. 10A and 10B and FIG. 11 shows the effects of Compound 2 in thediet-induced obesity model. At baseline, there was no significantdifference in body weight and daily food and water intake between the 3DIO groups. However, all DIO groups were significantly different fromthe regular diet group. Specifically, the animals fed under the regulardiet were of significantly lower body weight relative to rats fed thehigh fat diet. Conversely, rats fed the high fat diet consumedsignificantly less daily food and water relative to the rats fed theregular diet.

Compound 2 (1.5-3 mg/kg oral) produced a dose-related decrease in dailyfood intake and body weight over the 28 day treatment period compared tothe DIO control group. Compound 2 treatment also significantly increasedwater intake measured over the same period (F3,27=11.2, P<0.01).

In terms of treatment effect on body weight gain, this was formallymeasured as percentage of body weight change from study day 3. There wasa significant reduction in weight gain in both Compound 2 groupscompared to DIO controls at treatment days 7 (study day 10) and 14(study day 17).

Body weight, food/water intakes were measured daily over the washoutphase. Food intake in the Compound 2 groups was similar to DIO controls.Body weight in the Compound 2 groups remained lower than DIO controls.

Compound 2 decreases daily food intake in a dose dependent manner.Compound 2 also affects body weight gain at both 1.5 and 3 mg/kg doses.

Compound 1 Induction of the Nrf2 Anti-Inflammatory Pathway

THP-1 (human acute monocytic leukemia cells) cells were used to evaluatethe effects of Compound 1 on the Nrf2 pathway in an inflammationenvironment. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is ananti-inflammatory transcription factor. Under normal conditions, Nrf2 iskept in the cytoplasm by Kelch like-ECH associated protein 1 (KEAP1),which degrades Nrf2 by ubiquitination. Nrf2 can also move into thenucleus and back into the cytoplasm as a CRM1 cargo. In the currentstudy, Nrf2 was protected from degradation by knocking down KEAP1 withsiRNA. Then, KEAP1-depleted cells were treated with TNFα to induceinflammation, and the ability of Compound 1 to reverse inflammation byup-regulation of the Nrf2 pathway was tested. To demonstrate activationof the Nrf2 pathway, the expression of two of its downstream genesNAD(P)H dehydrogenase [quinone]1 (NQO1) and epoxide hydrolase 1 (EPHX1)were quantified by quantitative PCR.

THP-1 (acute monocytic leukemia) cells were plated in two 10 cm culturedishes (6*10⁶ cells/dish) with RPMI-1640 medium (Lonza) supplementedwith 10% heat-inactivated fetal bovine serum (Invitrogen) and2-mercaptoethanol to a final concentration of 0.05 mM. Cells in one dishwere transfected with 50 nM of KEAP1 siRNA (Life Technologies, SilencerSelect, siRNA ID# s18982) using Lipofectamine RNAiMax (Invitrogen),whereas the cells in the other dish were transfected with 50 nM ofcontrol siRNA, Block-iT (Invitrogen). Transfected cells were left for 72h and the KEAP1 knockdown efficiency was calculated with quantitativePCR using a probe against KEAP1.

Next, the cells from each of the dishes were divided equally into 4wells in different 6-well plates. One of the wells from each of theplates was pre-treated with 1 μM of Compound 1 for 1 h, followed by 20ng/mL TNFα for 24 h. The other wells were treated with either 1 μMCompound 1 or 20 ng/mL TNFα or neither for 24 h. Following thetreatment, RNA was extracted from the cells using an RNA extraction kit(Qiagen). RNA samples from each treatment group were reverse transcribedand real-time PCR was performed on the corresponding cDNA sequencesusing probes against Nrf2 and two of its downstream genes, NQO1 andEPHX1. THP-1 cells were transfected with KEAP1 siRNA. 40% knockdownefficiency was achieved. The KEAP1 knockdown cells were treated witheither 1 μM of Compound 1 or 20 ng/mL of TNFα or both together for 24 h.

FIG. 12A shows a 2.5-fold increase in Nrf2 expression in cells treatedwith a combination of TNFα and Compound 1 when compared to the untreatedcells. But, a similar (up to a 3-fold) increase in Nrf2 mRNA levels wasalso found in cells treated with Compound 1 and TNFα without the KEAP1knockdown. Compound 1 or TNFα alone did not have any significant effecton Nrf2 expression with or without KEAP1 knockdown.

FIG. 12B shows the expression of NAD(P)H dehydrogenase [quinone]1 orNQO1 in cells with or without KEAP1 knockdown. FIG. 12B shows that KEAP1knockdown had an effect on NQO1 expression. Even the sample without anytreatment showed a 2-fold increase in its mRNA levels upon KEAP1knockdown. The combination of Compound 1 and TNFα resulted in a 4-foldincrease in NQO1 expression for the KEAP1 knockdown sample compared to a2-fold increase, seen with the same combination in cells without KEAP1knockdown.

FIG. 12C shows the mRNA levels of epoxide hydrolase 1 or EPHX1 in cellswith or without KEAP1 knockdown after treatment with Compound 1 and/orTNFα. FIG. 12C shows that Compound 1 up-regulated the expression ofEPHX1 in the presence or absence of TNFα. KEAP1 knockdown added to theeffect of Compound 1, as induction up to 2.5-fold was observed in thesamples with Compound 1 and KEAP1 knockdown.

Treatment with 1 μM Compound 1 for 24 hrs in the presence of 20 ng/mLTNFα up-regulated Nrf2 signaling. KEAP1 knockdown enhanced this effect,as seen by larger fold induction of NQO1 (4-versus 2-fold) and EPHX1(2.5-versus 1.5-fold) relative to their levels of expression withoutKEAP1 knockdown. The results show that CRM1 inhibition can activate Nrf2pathway during inflammation, and suggests that treatment of Compound 1in combination with KEAP1 inhibitors could be more effective thantreatment with Compound 1 alone.

Effects of Compounds 1, 2, and 12 on NF-κB Transcriptional Activity

TNFα can induce the transcription activity of NF-κB. This transcriptionactivity is initiated when IκB, which binds to NFκB and inhibits itsactivity, is degraded. Then, a member of the class II family of NF-κBprotein, RelA or p65, that forms a heterodimer with a member of theclass I family, p50, moves into the nucleus. The p65 subunit has atransactivation domain in its C terminus, which activates transcriptionof inflammation related genes. Like NF-κB, IκB can also move into thecell nucleus. Nuclear accumulation of IκB protects the protein fromdegradation, as degradation occurs mainly in the cytoplasm. CRM1 isresponsible for the nuclear export of IκB. Therefore, blocking nuclearexport of IκB through inhibition of CRM1 minimizes NF-κB activity, asnuclear IκB binds NF-κB and prevents NF-κB from binding to DNAsequences.

The compounds were tested on HeLa (adenocarcinoma) cells to quantifytheir ability to inhibit NF-κB transcriptional activity. NF-κB activitywas induced in HeLa cells by TNFα, and then the compounds were added toinhibit the induced NF-κB activity. Half maximal inhibitoryconcentrations (IC₅₀) of several compounds, namely Compound 1, Compound2 and Compound 12, were determined by dose response studies.

HeLa cells were plated in a 12-well plate (200,000 cells/well) andcultured in Eagle's Minimal Essential medium (EMEM) from Lonzasupplemented with 10% heat-inactivated fetal bovine serum (Invitrogen)and 50 μg/mL penicillin/streptomycin (Invitrogen), and were leftovernight to attach. Cells were pre-treated with serial diluted (startedat 30 M; 1:3 dilution) compounds for 1 h and then exposed to 20 ng/mLTNFα (Peprotech) for 4 h in serum free media. After the treatment, thecells were washed with PBS (Invitrogen), and lysed with RIPA buffer(Themo Scientific). The transcription activity of NF-κB in the cells wasmeasured by Chemiluminescent Transcription Factor Assay kit (ThermoScientific Catalog#89859), according to the manufacturer's instruction.Briefly, 1.5 mg/mL of RIPA lysed whole cell extract from each treatmentwere incubated in a 96-well plate bound with NF-κBbiotinylated-consensus sequence. The active NF-κB transcription factorbound to the consensus sequence was incubated with NF-κB p65 primaryantibody and then with a secondary HRP-conjugated antibody. Achemiluminescent substrate was added to the wells and the resultingsignal was detected using a luminometer. Three separate experiments wereanalyzed for each concentration of the IC₅₀ curves. XLFit model 205 wasused to calculate IC₅₀ curves.

Inhibition of NF-κB transcriptional activity was measured by serialdilutions of Compound 1, Compound 2 and Compound 12 after 1 h ofcompound pre-treatment followed by 4 h of 20 ng/mL TNFα exposure. Threeindependent experiments were scored for each concentration, with theaverage being presented here. Compound 1 had an 1C₅₀ value of 1.59 μM,Compound 2 an IC₅₀ value of 1.22 μM, and Compound 12 an 1C₅₀ value of1.46 μM.

Evaluation of the Effects of Compound 1 on the Expression of thePro-Inflammatory Protein, COX-2, in HeLa Cells Grown In Vitro

HeLa cells were plated in a 6-well culture dish (2.5×10⁵ cells/well)with EMEM medium (Lonza) supplemented with 10% heat-inactivated fetalbovine serum (Invitrogen). Two of the wells of the plate werepre-treated with 10 μM Compound 1 for 30 minutes, at which time one oneof the wells was exposed to 20 ng/ml TNFα (Preprotech) for 1 hour. Theother wells were treated with either 20 ng/ml TNFα or nothing for 1hour. Following the treatment, RNA was extracted from the cells usingRNA extraction kit (Qiagen). RNA samples from each treatment group werereverse transcribed and quantitative real time (qRT) PCR was performedon the corresponding cDNA sequences using probes against COX-2 (LifeTechnologies).

HeLa cells were plated in a 6-well culture dish (5×10⁵ cells/well) withEMEM medium (Lonza) supplemented with 10% heat-inactivated fetal bovineserum (Invitrogen). Two of the wells of the plate were pre-treated with1 μM of Compound 1 for 30 minutes, at which time one of the wells wasexposed to 20 ng/ml TNFα (Preprotech) for 24 hours. The other wells weretreated with either 20 ng/ml TNFα or nothing for 24 hours. Following thetreatment, whole-cell lysates were generated from the cells by lysiswith RIPA buffer supplemented with protease and phosphatase inhibitors(Roche). Immunoblot detection of COX-2 protein was performed using ananti-COX-2 antibody (Cayman). Signal intensity for the COX-2 protein wasnormalized to that of beta-actin (Santa Cruz) for each sample andplotted graphically as arbitrary intensity units.

Data from the mRNA analysis by qRT-PCR is shown in FIG. 13A. After 1hour of treatment, TNFα induced an approximately 8-fold increase in theexpression of COX-2 mRNA compared to the control, whereas Compound 1alone had no effect on the level of COX-2 expression. Compound 1 was notthe cause of the increase in COX-2 mRNA expression.

Data from the protein analysis by immunoblot is shown in FIG. 13B. HeLacells were left untreated, treated with either 20 ng/ml TNFα or 1 μMCompound 1, or with 20 ng/ml TNFα and 1 μM Compound 1 for 24 hours, thenevaluated for the amount of COX-2 protein present by immunoblotdetection. COX-2 protein increased by 24 hours in TNFα-stimulated cellscompared to untreated control and to Compound 1 treated cells, whileCompound 1 decreased the amount of COX-2 protein in the presence ofTNFα. The intensity of the immunoblot signals for COX-2 protein werenormalized to that of β-actin for each sample and representedgraphically.

Compound 1 does not affect the TNFα induced expression of COX-2, butdoes reduce the amount of TNFα induced expression of COX-2 protein.

Compound 1 Localizes Inflammation-Related CRM1 Cargos to the Nucleus

HeLa and THP-1 (human acute monocytic leukemia) cells were treated withinflammation inducing factor, TNFα, alone or in combination with 1-10 μMof Compound 1 for 4-24 h, and then were analyzed by immunofluorescence(IF) for the nuclear localization of inflammation-related CRM1 cargoproteins: IκB, Nrf2, HMGB1, FoxP3, FOXO1a, RxRα, PPARγ and NFκB (p65subunit).

For the detection of IkB, Nrf2, RxRα and PPARγ localization, cells werepre-incubated with 10 μM Compound 1 for 30 minutes, followed byincubation with 20 ng/mL TNFα for 4 hrs in serum free media. Fordetection of HMGB1, FoxP3 and Foxo1A, cells were pre-incubated with 1 μMof Compound 1 for 2 h, followed by incubation with 20 ng/mL TNFα for 24hours. Cells were either fixed with 100% ice-cold methanol (MeOH) andpermeabilized/blocked with 0.1% Tween 20, 0.3 M glycine, and 1% BSA inPBS, or fixed with PFA (3% paraformaldehyde and 2% sucrose in PBS) andpermeabilized/blocked with 0.1% Triton-X100 and 1% BSA in PBS. IκB wasdetected by the primary rabbit monoclonal (E130) antibody from Abcam(ab32518); Nrf2 was detected by the primary rabbit polyclonal antibodyfrom Santa Cruz (sc722); RxR alpha was detected by the primary rabbitpolyclonal antibody from Santa Cruz (sc553); PPAR gamma was detected bythe primary rabbit monoclonal [E130] antibody from Cell Signaling(#2443); Foxo1A was detected by the primary rabbit monoclonal [C29H4]antibody from Cell Signaling (#2880); HMGB1 was detected by the primaryrabbit polyoclonal antibody from Abcam (ab18256); FoxP3 was detected bythe primary rabbit polyclonal antibody from Abcam (ab10563). The rabbitsecondary antibody, Alexa Fluor 488 (Invitrogen, A11008) was used forall the staining. Images were taken at 20× magnification.

Locking inflammation-related CRM1 cargos in the nucleus has adverseeffects on inflammation and, therefore, IF assays can serve asbiomarkers for anti-inflammatory effects of CRMI inhibitors.

IκB is the inhibitor of NFκB that induces the expression ofpro-inflammatory pathways. Because most IκB degradation occurs in thecytoplasm, its nuclear localization protects IκB from degradation andenables it to bind to nuclear NFκB, blocking the pro-inflammationactivity of NFκB. Nrf2 is a leucine zipper transcription factor thatinduces in the nucleus the expression of anti-inflammatory activity.HMGB1 is the high-mobility-group box 1 factor, and is usually boundtightly to chromatin. Upon active secretion or passive release fromdamaged cells, HMGB1 functions as a cytokine and induces thepro-inflammatory response. Locking HMGB1 in the nucleus prevents itspro-inflammatory effects. FoxP3, forkhead box P3, functions as a mastertranscription factor in the development and function of regulatory Tcells that possess immunosuppressive activity. FOXO1a is a transcriptionfactor capable of inducing anti-inflammatory genes, such asangiopoietin-2. Therefore, nuclear localization protects FOXO1a fromphosphorylation, nuclear exclusion and subsequent degradation. RxRα is aretinoid nuclear receptor that regulates the expression of chemokinessuch as Ccl6 and Ccl9 in macrophages. RxRα is essential for therecruitment of leukocytes to sites of inflammation. Nuclear entrapmentof RxRα results in the recruitment and the depletion of transcriptionco-activators that otherwise serve to bind pro-inflammatorytranscription factors such as NFκB. PPARγ is a ligand-activatedtranscription factor belonging to the nuclear receptor superfamily, andregulates the expression of anti-inflammation genes.

The results, shown in FIG. 14, demonstrate nuclear localization of theabove cargos and, therefore, indicate the ability of Compound 1 toinduce anti-inflammation pathways to overcome inflammation.

Evaluation of the Effect of Compound 1 on Cognitive Deficits after BCCIInjury in Rats

Bilateral controlled cortical impact (BCCI) injury to the medial frontalcortex (MFC) of male Sprague Dawley rats was induced by a corticalcontusion device. After CCI any cortical surface hemorrhaging wascontrolled, and the fascia and scalp were sutured. Sham-operated ratswere anesthetized, mounted in the stereotaxic apparatus, and acraniotomy was performed.

Progesterone 16 mg/mL was dissolved in 22.5%2-hydroxypropyl-3-cyclodextrin and the initial injection (16 mg/kg) wasgiven i.p. 1 h after injury. The remaining injections (all 16 mg/kg)were given subcutaneously at 6 h post-injury and continued for 5 daysafter injury. Progesterone injections were made at a concentration of 1mL/kg. Progesterone was used as a control.

Compound 1 0.2, 0.4, and 0.6 mg/mL was suspended in vehicle (0.6% w/vPluronic® F-68 and 0.6% w/v PVP K-29/32 in water) and administered p.oat a concentration of 10 mL/kg, 16 h before injury and 2 h after injury,and administrations were continued for 4 days. Control rats receivedequivalent injections of the vehicle for Compound 1, at the same timepoints. Treatment groups are summarized in Table 11.

TABLE 11 Treatment Groups Route of Number of Admin- Group animals TestArticle Dose istration Schedule 1 8 Vehicle + Sham N/A PO 16 h beforeinjury, 2 h after injury and continued for 4 days. 2 8 Vehicle + BCCIN/A PO 16 h before injury, 2 h after injury and continued for 4 days. 38 Progesterone 16 IP + SC 1 h after injury, [IP] 6 h post-injury mg/kgand continued for 5 days after injury [SC] 4 8 Compound 1 2 PO 16 hbefore injury, 2 h after injury mg/kg and continued for 4 days. 5 8Compound 1 4 PO 16 h before injury, 2 h after injury mg/kg and continuedfor 4 days. 6 8 Compound 1 6 PO 16 h before injury, 2 h after injurymg/kg and continued for 4 days.

The Morris Water Maze (MWM) test is a spatial navigation task thatmeasures learning and memory in rodents using visual cues. Subjectslearn over the course of days to find a hidden platform. A MWM test wasconducted two weeks after injury. Male Sprague Dawley rats were allowedto swim in the pool until they reached the platform located in thesouthwest quadrant of a tank, or until 90 seconds had elapsed. Behaviorwas tracked by a video camera hanging from above the pool and recordedand analyzed using video track software (ANY-maze).

The effects of Compound 1 and progesterone on acquisition of the MWMtest are shown in FIG. 15A. Two way repeated measures ANOVA found asignificant treatment effect. Compared to sham injury rats, BCCI-injuredrats showed a significant spatial learning deficit, as indicated by asignificant increase in the latency to find the hidden platform duringthe 5-day acquisition phase (FIG. 15A). Compared to vehicle-treatedBCCI-injured rats, Compound 1 (2, 4 and 6 mg/kg) showed a dose dependentdecrease in the latency to find the hidden platform, with significanteffects on days 17 and 18 after injury with 6 mg/kg and on day 18 with 4mg/kg. The data suggest that Compound 1 has a neuroprotective effect.

FIG. 15C is photographs of whole brains of animals receiving shamlesions (Sham), CCI+vehicle (Control), or CCI+Compound 1 (6 mg/kg), andshows the results of a qualitative visual inspection of whole brainsprior to vibratome sectioning. The inspection indicated that none (0 of4) of the Sham animals exhibited damage to dorsal-medial corticaltissue. In stark contrast, all four of the CCI controls exhibited severebilateral injury restricted to this region of the cortex. CCI animalswhich received Compound 1 showed damage ranging from moderate tominimal. Notably, the brain demonstrating the most severe injury in theCompound 1 group was less dramatic than all brains in the CCI controlgroup.

The expression level of several cytokines in plasma harvested from ratsin each treatment group was measured. The samples were received frozenand stored at −80° C. On the day of the experiment, the samples werethawed, diluted four-fold, and analyzed for cytokine expression on aLuminex platform. The samples were analyzed for the cytokines GRO/KC,IFNy, IL-1B, IL-6, IL-10, IL-12p70 and TNFα, using a multipliex kitmanufactured by Millipore. As shown in FIG. 15B, the same patterns wereobserved in expression levels between samples. The biggest change was inIL-10. Compound 1 at 6 mg/kg reduced IL-10 compared to vehicle-treatedcontrol group.

In many cases, traumatic injury elicits a secondary injury response. Inmost cases, the result will be inflammation. The inflammatory responseis driven by cytokines and chemokines and is partially propagated bydamaged tissue derived products (Damage associated Molecular Patterns).

Multiple Organ Dysfunction Syndrome (MODS), a poorly understood syndromeof sequential and gradual loss of organ function, is the most frequentcause of late deaths post-injury, accounting for substantial morbidityand mortality. MODS is considered to be due, in part, to excessive ormaladaptive activation of inflammatory pathways.

Quantitative measures of cell density were collected from anti-NeuNimmunolabeled subsections from approximately 2-3 mm anterior to bregma.Regions of interest (ROIs) were drawn (blind to experimental condition)around Layers IV-VI in the cortical region adjacent to the injury sitein CCI-treated animals or in the equivalent zone (dorsal cortex) in shamanimals. An ROI of similar area was also evaluated in a ventral corticalregion of the same section. Cell identification was performed using thecell counting module of Keyence BZ-II Analyzer software. Cell-to-graymatter (CG) area coefficients were determined for each ROI. Sham animalsexhibited uniform dense labeling within both dorsal and ventral regions.As expected, CCI control animals showed reduced CG coefficients in bothdorsal (−45% compared to sham) and ventral (−30% compared to sham)cortical zones versus sham animals (FIG. 15D). The CCI-induced reductionin CG coefficient was mitigated by treatment with Compound 1 in theventral cortex (−3% compared to sham; p=0.09). Although the effect ofCompound 1 versus vehicle treatment in the ventral cortex is notstatistically significant, it is anticipated that this effect wouldbreach statistical significance in a larger study. No effect of Compound1 was detected in the dorsal cortical region (−32% compared to sham)immediately adjacent to the injury site. The difference in the observedeffect of Compound 1 on the ventral region versus the dorsal region maybe a threshold effect related to the degree of injury, which wasinversely related to the distance from the injury site. Thus, the damageto the dorsal cortical region could be too severe to be rescued byCompound 1 under these conditions.

Immunofluorescence was performed to evaluate the impact of TBI onseveral pathways of immune response, and to determine if Compound 1might be mediating its neuroprotective effects via one or more of thosepathways. Semi-quantitative measures of secondary injury responses wereexamined using immunofluorescent labeling for anti-Rat IgG (an indicatorof blood-brain barrier (BBB) permeability), and TNFα (an indicator ofneural inflammation). All markers were imaged at 20× magnification inthe areas of the cortex surrounding the injury site in adjacentsubsections within 300 μm to those used for the NeuN labelingassessments. For each label, a target ROI was outlined within LayersIV-VI adjacent to the injury site (or an equivalent region of dorsalcortex for sham animals), and a reference ROI was collected from thesame laminae in ventral cortex. Normalized fluorescence intensity wasassessed in each of the two ROIs. For all labels, the ventral cortexreference site was determined not to be different between groups(p>0.5); therefore, the percent target to reference value (IF) wasdetermined.

Anti-Rat IgG was expressed in neurons (indicated by the arrowhead inFIG. 15E) in injured tissue. FIG. 15E shows that the anti-rat IgG wasdistributed within the neurophil of damaged areas of cortical tissue.Anti-rat IgG was not present in sham tissue. TNFα immunopositive cells(indicated by the arrowhead in FIG. 15E) were clearly visible in damagedtissue surrounding the injury site in control animals. These elementswere largely absent in Compound 1-treated and sham animals. FIG. 15Eshows that Compound 1 reduces secondary injury responses in rats exposedto brain injury.

Collagen-Induced Arthritis (CIA) Study No. 2

To further investigate the effect of the compounds described herein oninflammation biomarkers, a second CIA model was initiated. In thismodel, the groups were designated as group A (naïve), group B (model;vehicle-treated), group C (Compound 2 at 5 mg/kg QoD). The rats ingroups B and C were immunized intradermally with bovine Type II collagenin IFA on day 0, and a booster injection was given on day 7. Compound 2was orally administered to rats with CIA after the onset of arthritis(Day 11). CIA development was evaluated via macroscopic scoring andmeasurements of paw swelling. This was assessed every day for the first5 days after sensitization (day 7), and then twice per week (Monday andThursday) until Day 28 using the clinical scoring system described inTable 7 above. In addition, ELISAs for CD45, CRP, CCL2/MCP-1, RANKL,TNF-α, IL1-β, IL-6, IL-8, IL-15, IL-17, and measurements for cathepsin Kand elastase were performed 4 days (Day 15 of the study) and 10 days(Day 21 of the study—peak for the disease) after compound treatment andat the very end of the study (Day 28) on all group animals.Additionally, on the last day of the study (Day 28), a fewrepresentative animals from each group were subjected tothree-dimensional micro-tomodensitometry of calcaneus, and bone erosionwas quantified.

FIGS. 16A and 16B show that rats treated with Compound 2 at 5 mg/kg hadsignificantly reduced joint swelling (FIG. 16B) and clinical scores(FIG. 16A) compared to vehicle-treated rats.

FIGS. 17A and 17B show that rats treated with Compound 2 at 5 mg/kg hadsignificantly reduced bone erosion in the rear paws compared tovehicle-treated rats. Joint condition in animals treated with Compound 2was comparable to that of naïve animals. In contrast, vehicle-treatedanimals displayed statistically significant increased bone erosion intheir rear paws.

Experimental Autoimmune Encephalomyelitis (EAE) Model

The EAE Model is an accepted model for the study of human CNSdemyelinating diseases such as multiple sclerosis. The effects ofCompound 1 were investigated in MOG-induced in an EAE murine model infemale C57Bl/6J mice. The animals were divided into 3 groups designatedas Group I (vehicle control), Group II (dexamethasone-positive control)and Group III (Compound 1 at 7.5 mg/kg). Saline, dexamethasone andCompound 1 were administered according to the schedule shown in FIG.18A. Saline and dexamethasone were administered intraperitonally everyday starting from day 0. Compound 1 at 7.5 mg/kg was administered orallystarting from day 11 (disease onset) on Monday, Wednesday and Friday for3 consecutive weeks. The disease was induced by the single intradermalinjection of MOG emulsified in Complete Freund's Adjuvant (CFA) on studyday 0, followed by intraperitoneal supplemental immunostimulation withpertussis toxin (PT) carried out on study day 0, and again 48 hourslater on study day 2.

As shown in FIG. 18B, the first signs of the disease were noticed 7-9days following MOG immunization and the disease peak developed on studyday 17. Treatment with dexamethasone starting from day 0 at a dose of 1mg/kg IP significantly reduced the clinical scores on study days 8-37(Group II) when compared to the vehicle control (Group I). Treatmentwith Compound 1 starting from Day 11 at a dose of 7.5 mg/kg (Group III)significantly decreased disease score and severity. These results areshown in FIG. 18B.

In view of the findings obtained under the conditions of this study,treatment with Compound 1 at a dose of 7.5 mg/kg p.o. starting on studyday 11 resulted in a decrease in disease score and severity.

Example 18. Wound Healing Models Materials

Mice—C57BL/6J mice, males, aged 6-8 weeks, SPF, obtained from HarlanLaboratories LTD. Mice were kept in sterile individual ventilated cages(IVC) with food and water available ad libitum, 12 h/12 h cycles ofdarkness and light, controlled temperature of 19-21° C., controlledhumidity of 40-60%, positive air pressure inside animal's room, andhealth report control every 3 months, which was performed on selectedsentinels.

Pigs—sus scrofa domestica, Domestic swine (mainly Landrace×large White),female, approximately 60 Kg, 4-5 months old, Lahav Institute of AnimalResearch, Kibbutz Lahav, Israel. Pigs kept in clean non-SPF environment,tap water ad libitum directly from public source, food according torecommendation of standard growth tables under supervision ofveterinarian.

ISOFLURANE 99.9% for inhalation, lot 6027962, Abbot Laboratories Ltd,England

Water—water for injection, batch 11481012, B. Braun Melsungen AG,Germany

Saline—0.9% sodium chloride for injection, batch 12224012, B. BraunMelsungen AG, Germany

DMSO—dimethyl sulfoxide, D2650, Sigma-Aldrich Inc., U.S.

PLURONIC® F-68

PVP K-29/32

Evaluation of the Effects of Systemic Administration and TopicalApplication of Compound 1 on C57BL Mice Skin Wounds

The effects of Compound 1 on skin wound healing were studied in a mouselongitudinal full thickness skin incision wound model. Upon arrival,animals were identified by ear tags, weighed and left to acclimate forseveral days before initiation of the experiment. On the day ofwounding, mice were weighed and divided into 6 experimental groups with6 animals per group, in accordance to weight differences stratifiedrandomization. Prior to the surgical procedure, mice were anesthetizedwith isoflurane and the back of the animals was trimmed. Full thicknesslongitudinal incisions of 20 mm were performed using a standard scalpelblade on the backs of the animals (parallel to the backbone). Threehours after wounding, due to skin elasticity and activity of theanimals, the incisions took on elliptical shapes. At this stage, thewidest area of the wound was measured to establish a baseline woundwidth. Wound healing evaluation was made by measuring the widest area ofthe wound. Treatment groups consisted of oral gavage or topical groups.During the experiment, wounds were photo-documented and morphologicalanalysis was performed. At the end of the experiment, 8 days postwounding, mice were sacrificed, wound widths were measured and biopsiesof the wound area were collected and subjected to analysis.

TABLE 12 Initial Study Groups Route of Number Admin- Group of mice TestArticle Dose istration Schedule 1 6 Control aqueous 0.2 mL PO ever 0.6%w/v other Pluronic ® F-68 and day 0.6% w/v PVP K- 29/32 solution 2 6Compound 1 in   4 mg/kg PO every PVP/Pluronic ® other F-68 day 3 6Compound 1 in 7.5 mg/kg PO every PVP/Pluronic ® other F-68 day 4 3Vehicle, water for 0.2 mL Topical Daily injection 5 6 Compound 1 in 2.5μM Topical Daily water 6 6 Compound 1 in   1 μM Topical Daily water

Dosing solutions were prepared fresh on each day of dosing. Compound 1for oral gavage was supplied as a lyophilized powder and reconstitutedin aqueous 0.6% w/v Pluronic® F-68 and 0.6% w/v PVP K-29/32 solution tomake a 0.75 mg/mL stock suspension, which was subsequently diluted withaqueous 0.6% w/v Pluronic® F-68 and 0.6% w/v PVP K-29/32 for preparationof working solutions of 7.5 mg/kg and 4 mg/kg. Compound 1 for topicalapplication was supplied as a lyophilized powder and suspended in waterto a concentration of 10 mM, which was further diluted with water toachieve a final working concentration for topical application.

As a part of a daily morphological assessment, photo-documentation wasperformed using a digital camera FinePix S700. FIG. 19 is photographs ofrepresentative wounds from each experimental group on Day 5post-wounding. The black scale bar represents 1 cm. A total of 33 woundswere made in 33 mice. The morphological assessment demonstrated thepositive effect of treatment with Compound 1, either orally ortopically. All treatments induced superior wound healing than controls.Treated wounds were smaller in size and the scabs were lighter, thinnerand homogenous without cracks, indicating a later stage of woundhealing. When evaluated on the same day as treatment groups, controlgroup wounds appeared larger in size and were covered with thick crackedscabs that exposed a non-healed wound area (observed as reddish and pinkareas) both at the edges and in the middle of the wounds.

Morphological analysis is the primary parameter utilized in woundhealing assessment in preclinical studies on animals and in clinicaltreatments of human wounds. Based on morphological analysis, Compound 1displayed efficacy, and had a positive impact on wound healing. Of note,both topical application and systemic administration of Compound 1resulted in better wound healing, as measured by wound size reductionand better scabbing properties.

Evaluation of the Effects of Topical Application of a Test Compound onPig Skin Wounds

The effects of a test compound on skin wound healing can be studied in apig longitudinal full thickness skin incision wound model. Upon arrival,animals are identified by ear tags, weighed and left to acclimate forseveral days before initiation of the experiment. Three days prior tothe surgery, pigs are transferred to the hospitalization facility foracclimation. Twelve hours prior to the procedure, food is withheld. Onthe day of surgery, the pig is anesthetized using ketamine, xylazin,diazepam and isoflurane. The hair on the dorsum thorax and abdomen iscarefully cut using an Oster® clipper machine (blade size 30) and 20individual regions of 4 cm² each are marked in two rows (10 regions perrow). Ten pairs of 2.5 cm full thickness longitudinal skin incisions aremade using #11 scalpel blade, 4 cm from either side of the dorsummidline.

Following the surgical procedure, wounds are divided into experimentalgroups and treated daily by topical application on the wound area and onwound edges. Treatment area consists of a surface of skin up to adistance of 2 cm from the wound center. Dosing solutions are appliedgradually on each wound using a pipette, until the entire treatmentvolume (for example, 1 mL of saline or test compound) is absorbed bytissue.

Several hours after wounding, due to skin elasticity and activity of theanimals, the incisions take on elliptical shapes. At this stage, thewidest area of the wound is measured to establish a baseline woundwidth. Wound healing evaluation is made by measuring the widest area ofthe wound. During the experiment, wounds are photo-documented andmorphological analysis is performed.

At the end of the experiment (for example, 12 days after wounding), pigsare sacrificed by administration of anesthetic and KCl. Wound morphologyis assessed, wound width is measured and biopsies of wound area areharvested and fixed using 4% paraformaldehyde for further analysis.Following fixation, wound biopsies are photo-documented using highresolution digital camera, for example, a FinePix S700, and biopsies ofthe wound area are subjected to histopathological analysis. Assessmentof wound healing is performed in a paired manner in which each woundtreated with test compound is directly compared to the control wound atthe same anatomical location on the other side of the dorsum midline.This paired assessment of healing is crucial in terms of objectiveassessment and objective comparison of treated wounds to non-treatedbecause of variability associated with a degree of vascularization andblood circulation in the skin at different areas of the pig's back.Wounds located in the front area near the neck display far betterhealing properties than wounds located on the rear back.

TABLE 13 Initial Study Groups Route of Number of Admin- Group woundsTest Article Dose istration Schedule 1 5 front wounds Control saline 1mL Topical Daily on the right side 2 5 rear wounds Control saline 1 mLTopical Daily on the right side 5 3 5 front wounds Test compound 3 μMTopical Daily on the left side 4 5 rear wounds Test compound 1 μMTopical Daily on the left side 5

Dosing solutions are prepared fresh on each day of dosing. Test compoundis supplied as a lyophilized powder and further reconstituted ininjectable 0.9% sodium chloride to make a 3 mg/mL stock suspension. Thestock suspension is further diluted with injectable 0.9% sodium chlorideto final concentrations of 3 μM and 1 μM for topical application.

Homogenous, thin and uniformly organized scab surfaces without incidentsof oozing, bleeding or secretion from the wound are indicative of woundhealing. Highly heterogeneous, cracked and dark colored scabs indicatenumerous incidents of exudation, oozing and bleeding during the courseof the wound healing process.

Evaluation of the Effects of Topical Application of a Test Compound onEarly Wound Healing Processes in Pigs

The effects of a test compound on early wound healing can be studied ina wound model of longitudinal full thickness skin incision in pigs. Fivepairs of 2.5 cm longitudinal full thickness incisions are performed onthe frontal section of the back of anaesthetized pigs using #11 scalpelblades, 4 cm from either side of the dorsum midline. Within severalhours post-procedure, the longitudinal incision becomes an ellipticalwound.

Wounds are divided into experimental groups and treated daily by topicalapplication on the wound area (including edges and on skin area near thewound). Treatment phase starts 24 hours following wounding. Dosingsolutions are applied gradually on each wound using a pipette, until theentire treatment volume is absorbed by tissue (for example, 1 mL ofsaline or a test compound).

On day 5, the state of wound healing and morphology is assessedaccording to the following parameters: bleeding, oozing, swelling,inflammation, pus secretion and scab formation. Assessment is performedin a paired manner in which each wound treated with the test compound isdirectly compared to the control wound at the same anatomical locationon the other side of the dorsum midline.

TABLE 14 Initial Study Groups Route of Number of Admin- Group woundsTest Article Dose istration Schedule 1 5 Control saline 1 mL TopicalDaily 2 5 Test compound 3 μM Topical Daily

Dosing solutions are prepared fresh on each day of dosing. Test compoundis supplied as a lyophilized powder and reconstituted in 0.9% sodiumchloride to a 3 mg/mL stock suspension. This stock suspension is furtherdiluted with 0.9% sodium chloride for the preparation of the final 3 μMtopical solution.

A morphological wound healing assessment is conducted on Day 5 oftreatment. Swelling is examined, scored according to the severity ineach wound and documented as mild, moderate or severe. Wounds thatexhibit moderate and severe swelling are presented as a percentage oftotal wounds in experimental group. Secretion is examined and scored ina binary mode: a wound that exhibited minimal secretion was consideredpositive and, a wound without any detectable secretion is considerednegative for this parameter. Wounds that exhibit secretions (positivefor this parameter) are presented as a percentage of total wounds inexperimental group. A scab is considered completely formed when acontinuous layer of a hard, dry, reddish, dark yellow or brown formationcovered the entire wound area and is strongly attached to the wound bedand, therefore, provided a continuous and strong barrier between theexternal environment and the wounded tissues. Scab formation is examinedand scored in a binary mode: wounds which exhibited a completely formedscab which was dry and strong are considered as positive and woundswithout a scab or with scabs at an earlier stage are considered asnegative for this parameter. Wounds with a completely formed scab arepresented as a percentage of total wounds per group.

Swelling, secretion and scab formation are also evaluated. Swelling andsecretion are part of an excessive inflammatory response that mightdelay tissue repair and induce unaesthetic scarring.

Evaluation of the Effects of Topical Application of a Test Compound onEarly Wound Healing on Pig Skin and on Irritations and ScratchingAssociated with Damaged or Wounded Skin

The effect of a test compound on skin wound healing can be studied in alongitudinal full thickness skin incision wound model in pigs. Threedays prior to surgery, pigs are transferred to the hospitalizationfacility for acclimation. Twelve hours prior to the surgical procedure,food is withheld. On the day of surgery, the pigs are anesthetized usingketamine, xylazin, diazepam and isoflurane. The hair on the dorsumthorax and abdomen is cut using Oster® clipper machine (blade size 30).Ten pairs of 4 cm² each sections are marked, and 2.5 cm full thicknesslongitudinal skin incisions are made using #11 scalpel blade, on eitherside of the dorsum midline.

Following surgical procedure, wounds are divided into experimentalgroups and are treated daily by topical application on the wound area(including edges and on skin area near the wound up to a distance of 2cm from the wound in all directions). Dosing solutions are appliedgradually on each wound using a pipette, until the entire treatmentvolume is absorbed by tissue (for example, 1 mL of vehicle or testcompound).

Within several hours post-procedure, the longitudinal incision becomesan elliptical wound due to skin elasticity and activity of the animals.During the experiment, wounds are photo-documented and morphologicalanalysis is performed. Assessment of wound healing is performed in apaired manner in which each wound treated with test compound is directlycompared to the control wound at the same anatomical location on theother side of the dorsum midline. During the first 5 days followingwounding, wound morphology and animal behavior are recorded.

TABLE 15 Initial Study Groups Route of Number of Admin- Group woundsTest Article Dose istration Schedule 1 5 front wounds 0.02% DMSO 1 mLTopical Daily on the right side in water 2 5 rear wounds 0.067% DMSO 1mL Topical Daily on the right side in water 3 5 front wounds Testcompound 3 μM Topical Daily on the left side in 0.02% DMSO 4 5 rearwounds Test compound 1 μM Topical Daily on the left side in 0.067% DMSO

Dosing solutions are prepared fresh on each day of dosing. Test compoundis supplied as a lyophilized powder and dissolved in 100% DMSO to astock concentration of 15 mM. Further dilutions in injectable water areperformed to achieve a final concentration of 3 μM and 1 μM for topicalapplication.

As part of the daily morphological assessment, photo-documentation ofthe wounds is performed using, for example, a digital high resolutioncamera FinePix S700. In addition to the wound status, areas of irritatedand scratched skin are observed. Usually, the scratching does not causedamage to the wounds or interfere with the wound healing process becausethe wound is inflicted on the back near the dorsum midline, such that itis hard and almost impossible for the animal to reach the wounds.

Evaluation of the Effects of Compound 1 in in PVP/Pluronic® F-68 andCompound 1 in Water on Scratching Associated with Skin Healing in Mice

In the skin wound studies described herein in mice, the behavior of theanimals was also observed, and attempts to remove scars, signs ofdiscomfort, and scratching of the wound area were quantified. Abnormalbehavior and abnormal displays of scratching and signs of pain from allthe studies performed in mice were analyzed. In these studies, treatmentwas performed using Compound 1 in PVP/Pluronic® F-68 and Compound 1 inwater, and the respective vehicle controls.

During all wound healing experiments in mice, monitoring of healingparameters associated with wound healing, signs of skin irritations andother skin conditions at the area near the wound and the treated skinarea, was performed. Additionally, during the treatment phase of allskin healing models, special attention was paid to the behavior of theanimals, such as signs of discomfort and pain; and signs of scratchingand tampering with wounds and skin. Soothing and calming effects of thetreatment compounds were highly obvious in comparison to controlanimals, which were predisposed to tamper with their wounds.

In mice, treatment with Compound 1 in PVP/Pluronic® F-68 or Compound 1in water reduced the incidence of tampering with wounds in comparison tothe vehicle treated mice (DMSO in water, saline, PVP/pluronic or water).

In mice, tampering with wounds usually resulted in the removal of thescab and bleeding or damage to the newly formed tissue on the wound bedthat was strongly attached to the scab. The vast majority of suchincidents happened in vehicle treated groups (about 20-30% in allexperiments).

According to the summary of skin conditions and animal behavior, it canbe concluded that treatment of wounds with Compound 1 in PVP/Pluronic®F-68 or Compound 1 in water prevented tampering with wounds in mice,possibly, due to some soothing and calming effects of the treatmentcompounds on wounded and irritated skin.

Dose Response of a Test Compound on Skin Wound Healing in Mice

The effects of a test compound on skin wound healing can be studied inmice longitudinal full thickness skin incision wound model. Uponarrival, animals are identified by ear tags, weighed and left toacclimate for several days before initiation of the experiment. On theday of wounding, mice are weighed and divided into 7 experimental groups(N=6 or N=7), in accordance to weight differences stratifiedrandomization. The vehicle group receives 0.1% DMSO in water while thepositive control group is treated with an aqueous 0.6% w/v Pluronic®F-68 and 0.6% w/v PVP K-29/32. Prior to the surgical procedure, mice areanesthetized with isoflurane and the hair on the back of the animals istrimmed. Full thickness longitudinal incisions of 20 mm are performedusing a standard scalpel blade on the backs of the animals (parallel tothe backbone). Three hours after wounding, due to skin elasticity andactivity of the animals, the incisions take on elliptical shapes. Atthis stage, the widest area of the wound is measured to establish abaseline wound width. Wound healing evaluation is made by measuring thewidest area of the wound. Treatment of wounds is performed by topicalapplication (daily) of dosing solutions (for example, 0.2 mL) directlyon wounds.

TABLE 16 Initial Study Groups Route of Number Admin- Group of mice TestArticle Dose istration Schedule 1 7 0.1% DMSO in 0.2 mL Topical Dailywater 2 6 Test compound in   9 μM Topical Daily 0.1% DMSO 3 6 Testcompound in   3 μM Topical Daily 0.1% DMSO 4 6 Test compound in   1 μMTopical Daily 0.1% DMSO 5 6 Test compound in 0.3 μM Topical Daily 0.1%DMSO 6 7 Control aqueous 0.2 mL Topical Daily 0.6% w/v Pluronic ® F-68and 0.6% w/v PVP K-29/32

Dosing solutions are prepared fresh on each day of dosing. Test compoundis supplied as lyophilized powder and reconstituted in 0.1% DMSO inwater to a 3 mg/mL stock suspension. The stock suspension is furtherdiluted with 0.1% DMSO in water to prepare the final topical solution.Wounds in control groups are topically treated with 0.1% DMSO in water,or aqueous 0.6% w/v Pluronic® F-68 and 0.6% w/v PVP K-29/32 solution.

At the end of the experiment, 8 days post wounding, mice are sacrificedby inhalation of CO₂, wound widths are measured and biopsies of thewound area are collected and subjected to histological analysis. Thebiopsies are fixed using 4% paraformaldehyde. Following fixation of theentire wound area, a dissection of 5 mm of the widest area of the woundis performed and these specimens are subjected to paraffin embedding.Paraffin blocks are prepared utilizing standard procedures of graduatedehydration and paraffin embedding of tissues. Thereafter, histologicalsections are prepared and tissues are stained with hematoxylin and eosin(H&E) stain. H&E stained slides are examined and assessment of woundhealing efficacy is performed.

Advanced dermal closure is assessed on Day 8 by the examination of eosinstained healthy dermis and the newly formed dermis edges at the woundgap. Wounds with both dermal edges observed in 100× magnification fieldof the microscope (BX41 Olympus or Axiovert 25, Zeiss) are consideredpositive for the advanced dermal closure healing parameter. The numberof wounds with advanced dermal closure is presented as a percent oftotal wounds in experimental groups.

Advanced epidermal closure is assessed on Day 8 using H&E staining byanalyzing histological section at the widest area of the wound. Woundsthat exhibit the presence of a continuous layer of epidermis coveringthe entire wound gap and wounds with the most advanced migration of theepidermal edges observed in the microscope field at 400× magnificationare considered positive to advanced epidermal closure parameter. Theresults are presented as a percent of total per experimental group.

Epidermal migration is assessed on Day 8 using H&E staining by analyzingcondensed hematoxylin stained newly formed epidermis at both woundedges. The epidermal edge is considered migratory when newly formedepidermal edge covered about 20-30% of the wound gap. Migratoryepidermal edges in the groups are counted and presented as a percent oftotal number of epidermal edges (twice the number of wounds in thegroup). Both epidermal edges are considered migratory in wounds thatexhibited complete or advanced epidermal closure. A total of 62 woundsare made in 62 mice.

Treatment of Wounds with a Test Compound Prevents Wound HealingComplications, Such as Hyperplasia of the Epidermis and Adhesions

The effects of a test compound on skin wound healing can be studied in amice longitudinal full thickness skin incision wound model. Prior to thesurgical procedure, mice are anesthetized with isoflurane, and the backof the animals is shaved. Full thickness longitudinal incisions of 20 mmare performed using a scalpel blade on the backs (parallel to backbone)of the animals. Three hours after wounding, due to skin elasticity andactivity of the animals, the incisions take on elliptical shapes. Woundhealing evaluation is made by measuring the widest area of the wound.Treatment of wounds is performed by topical daily application of, forexample, 0.2 mL of test compound directly on the wounds. Wound careprocess is partially in a moist environment—after each daily treatment,wounds are wet for some time. At the end of the experiment, 8 days postwounding, mice are sacrificed, wound widths are measured and biopsies ofthe wound area are collected and subjected to histological analysis.

TABLE 17 Initial Study Groups Route of Number Admin- Group of mice TestArticle Dose istration Schedule 1 7 0.1% DMSO in 0.2 mL Topical Dailywater 2 6 Test compound   3 μM Topical Daily in 0.1% DMSO 3 6 Testcompound   1 μM Topical Daily in 0.1% DMSO 4 7 Control aqueous 0.2 mLTopical Daily 0.6% w/v Pluronic ® F-68 and 0.6% w/v PVP K-29/32

Dosing solutions are prepared fresh on each dosing day. Test compound issupplied as a lyophilized powder and reconstituted in 0.1% DMSO in waterto a 3 mg/mL stock suspension, which is subsequently diluted with 0.1%DMSO in water to achieve a working concentration for topicalapplication. Wounds in control groups are topically treated with 0.1%DMSO in water or aqueous 0.6% w/v Pluronic® F-68 and 0.6% w/v PVPK-29/32.

At the end of treatment phase, on day 8 post-wounding, mice aresacrificed by inhalation of CO₂ and biopsies of the wound area areharvested. Fixation of wound tissues is performed using 4%paraformaldehyde. Following fixation of the entire wound area, adissection of 5 mm of the widest area of the wound is performed andsubjected to paraffin embedding. Paraffin blocks are prepared usingstandard procedures of graduate dehydration. Thereafter, histologicalsections are prepared and tissues are stained with hematoxylin and eosin(H&E). Wound healing parameters are assessed and graphed.

Hyperplasia of the epidermis is assessed on Day 8. Non-migratory andhyperplastic epidermal edges in the group are counted, and are presentedas a percent of total number of epidermal edges (twice the number ofwounds in the group). Hyperplastic epidermal edges are assessed usingH&E staining by analyzing condensed hematoxylin stained areas of theepidermis. When the epidermal edge appears thicker than normal epidermisin a healthy skin area and when such an epidermal edge does not exhibitmigration toward sealing the wound gap, it is considered to behyperplastic and non-migratory.

Adhesions at the wound gap are assessed on on Day 8. Adhesions areassessed by analyzing cellular and tissue structures at the wound gap.The wound adhesions are scored on a mild, moderate or severe scale. Anegative score is considered when there is a clot at the wound gap ornormal granulation tissue is replaced by other tissue, such as skeletalmuscles or extensive lymphoid tissues. Several adhesions or abnormalgranulation occupying more than 40% of the wound gap area are consideredas severe. Adhesion is considered mild when it is non-significant anddoes not interfere with normal skin tissue renewal. Wounds with severeadhesions are calculated as a percent of total wounds per experimentalgroup and graphed as shown. A total of 32 wounds (64 epidermal edges)are made in 32 mice.

One of the most important wound healing complications is hyperplasia ofthe epidermis. As a response to the stress signals associated withwounding, proliferation of cells in the basal layer of the epidermisoccurs to compensate for skin loss. Normally, in uneventful woundhealing, epidermal cells initiate migration toward sealing the wound gapsoon after proliferation. When migration does not occur or is sloweddown, for example, in skin complications caused by hyperglycemia indiabetic wounds, epidermal hyperplasia becomes prominent, and may causeeven more complications in wound healing. In acute open wounds, as inthe model employed in this experiment, or in acute sutured wounds, suchas post-surgical wounds, a decline in epidermal healing associated withhyperplasia of epidermal edges increases risk for contamination andother wound healing complications such as wound dehiscence, fluiddraining from the wound, or tissue protruding from the wound.

In an effective wound healing process, the primary blood clot undergoesgradual changes in order to form granulation tissue at the wound gap,which, following remodeling, eventually becomes newly formed skin tissuewith fully restored functions. When adhesion of non-skin related tissuesoccurs in the wound gap, granulation tissue does not form properly and,as a result, final tissue remodeling is limited. This may cause furtherlimitations in the functions of healed skin.

Treatment of Wounds with a Test Compound in a Saline-Based FormulationImproves Wound Healing and Prevents Severe Adhesions

The effects of a test compound on skin wound healing were studied in amouse longitudinal full thickness skin incision wound model. Surgicalprocedures are performed on 7-8 weeks old C57BL male mice anesthetizedwith isoflurane. Prior to surgical procedure, mice are anesthetized withisoflurane and the fur is cut. Full thickness longitudinal incisions of20 mm are performed using a standard scalpel blade. Three hours afterwounding, due to skin elasticity and activity of the animals, theincisions take on elliptical shapes. At this stage, the widest area ofthe wound is measured to establish a baseline wound width. Wound healingevaluation is made by measuring the widest area of the wound. Treatmentof wounds is performed by a daily application of a topical 0.2 mLsolution directly on the wound. The wound care process is conductedpartially in a moist environment because after each daily treatment,wounds are wet for some time (3-5 hours). At the end of the experiment,8 days post wounding, mice are sacrificed, wound widths are measured andbiopsies of the wound area are collected and subjected to histologicalanalysis.

TABLE 18 Initial Study Groups Route of Number Admin- Group of mice TestArticle Dose istration Schedule 1 5 Saline 0.2 mL Topical Daily 2 7 Testcompound in   3 μM Topical Daily saline

Dosing solutions are prepared fresh on each dosing day. Test compound issupplied as a lyophilized powder and reconstituted in saline to make a 3mg/mL stock suspension, which is subsequently diluted with saline toachieve a working concentration of 3 μM for topical application. Woundsin control groups are topically treated with saline.

At the end of the treatment phase, 8 days post-wounding, mice aresacrificed by inhalation of CO₂ and biopsies of wound area areharvested. Fixation of wound tissues is performed using 4%paraformaldehyde. Following fixation of the entire wound area, adissection of 5 mm of the widest area of the wound is performed and thedissected area is subjected to paraffin embedding. Paraffin blocks areprepared using standard procedures of graduate dehydration. Thereafter,histological sections are prepared and tissues are stained withhematoxylin and eosin (H&E). Wound healing parameters are assessed andgraphed.

Epidermal closure is assessed using H&E staining by analyzinghistological sections at the widest area of the wound. Wounds whichexhibit the presence of a continuous layer of epidermis covering theentire wound gap, and wounds with the most advanced migration of theepidermal edges when both edges were observed in the microscope field at400× magnification are considered positive for the advanced epidermalclosure parameter. The results are presented as a percent of total perexperimental group.

Dermal healing is assessed by the examination of eosin stained healthydermis and the newly formed dermis edges at the wound gap. Wounds withboth dermal edges observed in 100× magnification field of the microscope(BX41 Olympus or Axiovert 25, Zeiss) are considered positive for theadvanced dermal closure healing parameter. The number of wounds withadvanced dermal closures is presented as a percent of total wounds inexperimental groups.

Granulation tissue is assessed utilizing H&E staining. When the primaryfibrin clot is replaced by fibrous connective tissue containingadipocytes, new capillaries and an infiltrate containing lymphoid cells,macrophages, and plasma cells the granulation tissue is consideredearly. Early granulation tissue replaced by tissue with a high abundanceof fibroblasts and collagen fibers is considered advanced. Overall,areas of advanced granulation tissue at the wound gap are documented aspercent of the total wound gap area. A wound gap displaying advancedgranulation tissue formation covering 40% of the wound gap is consideredpositive for this parameter. Results are calculated as a percent oftotal wounds per group.

Adhesions are assessed by analyzing cellular and tissue structures atthe wound gap. The wound adhesions are scored on a mild, moderate orsevere scale. A negative score is considered when there is a clot at thewound gap or normal granulation tissue is replaced by other tissue, suchas skeletal muscles or extensive lymphoid tissues. Several adhesions orabnormal granulation occupying more than 40% of the wound gap area areconsidered as severe. Adhesion is considered mild when it isnon-significant and does not interfere with normal skin tissue renewal.Wounds with severe adhesions are calculated as a percent of total woundsper experimental group. Nineteen wounds (38 epidermal edges) were madein 19 mice.

Evaluation of the Effects of Topical Application of a Test Compound onHealing Process and Scarring in the Late Stages of Wound Healing on PigSkin

The effects of a test compound on late stages of skin wound healing arestudied in a pig wound model of longitudinal full thickness incision. Onthe day of surgery, the pig is anesthetized using ketamine, xylazin,diazepam and isoflurane. The hair on the dorsum thorax and abdomen iscut and 10 pairs of 2.5 cm full thickness longitudinal skin incisionsare performed using a #11 scalpel blade, 4 cm from either side of thedorsum midline. Following the surgical procedure, wounds are dividedinto experimental groups and treated daily by topical application on thewound area and on wound edges including treatment of skin near the woundarea up to a distance of 2 cm from the wound in all directions. Dosingsolutions are applied gradually on each wound using a pipette, until theentire treatment volume (for example, 1 mL of vehicle or test compound)is absorbed by the tissue. The skin near the wound is treated with gauzesoaked in test compound or vehicle solution.

During the experiment, wounds are photo-documented and morphologicalanalysis is performed. At the end of the treatment phase (day 19post-wounding), pigs are sacrificed by dosing of anesthetic and KCl.Morphology of the wounds is examined, wounds are photo-documented andbiopsies of wound area are harvested for fixation and furthermorphological and histological analysis.

TABLE 19 Initial Study Groups Route of Number of Admin- Group woundsTest Article Dose istration Schedule 1 5 front wounds 0.02% DMSO 1 mLTopical Daily on the right side in water 2 5 rear wounds 0.067% DMSO 1mL Topical Daily on the right side in water 3 5 front wounds Testcompound 3 μM Topical Daily on the left side in 0.02% DMSO 4 5 rearwounds Test compound 1 μM Topical Daily on the left side in 0.067% DMSO

Dosing solutions are prepared fresh on each day of dosing. Test compoundis supplied as a lyophilized powder and dissolved in 100% DMSO toprepare a stock solution of 15 mM. Subsequently, dilutions in injectablewater are performed to achieve final concentrations of 3 μM and 1 μM fortopical application.

At the end of the treatment phase (day 19 post-wounding), the assessmentof wound healing is performed. Fully healed wounds are reported as apercent of total wounds per group. The average width of scars in thewounds that healed completely and exhibited full scab detachment is alsoreported. Scars were measured (mm) and the average width of scars andstandard deviation are calculated. A total of 20 wounds was performed.

At the end of the treatment phase (day 19 post-wounding), pigs aresacrificed by an overdose of anesthetic and KCl and biopsies of woundarea are harvested. Fixation of wound biopsies is performed using 4%paraformaldehyde. Following fixation, wound biopsies arephoto-documented using, for example, a digital camera FinePix S700 atthe highest resolution.

BIBLIOGRAPHY

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The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selected from hydrogen and C₁-C₄ alkyl; R² is selected from O and S; and R³ is selected from —N(R⁴)—(C₃-C₆ cycloalkyl), —C₁-C₆ alkyl, —(C₀-C₄ alkylene)-heterocyclyl, and —(C₀-C₄ alkylene)-heteroaryl, wherein any alkyl, alkylene, heterocyclyl, and heteroaryl portion of R³ is optionally and independently substituted; and R⁴ is selected from hydrogen and C₁-C₄ alkyl.
 2. The compound of claim 1, wherein, R¹ is selected from hydrogen and methyl.
 3. The compound of claim 2, wherein R¹ is hydrogen.
 4. The compound of claim 1, wherein R² is O.
 5. The compound of claim 1, wherein R⁴ is hydrogen.
 6. The compound of claim 1, wherein R³ is selected from —N(R⁴)—(C₃-C₆ cycloalkyl), —C₃-C₆ alkyl, —(C₀-C₁ alkylene)-heterocyclyl, and —(C₀-C₁ alkylene)-heteroaryl, wherein: any alkyl or alkylene portion of R³ is optionally and independently substituted with one or more substituents selected from the group consisting of oxo and —N(R⁵)₂, wherein each R⁵ is independently selected from hydrogen and C₁-C₄ alkyl; any heterocyclyl portion of R³ comprises at least one nitrogen atom in a ring, and is optionally substituted with one or more substituents selected from the group consisting of C₁-C₄ alkyl and oxo; and any heteroaryl portion of R³ comprises at least one nitrogen atom in a ring and is optionally substituted with one or more C₁-C₄ alkyl.
 7. The compound of claim 6, wherein R³ is —(C₀-C₁ alkylene)-heterocyclyl.
 8. The compound of claim 7, wherein R³ is —(C₁ alkylene)-heterocyclyl.
 9. The compound of claim 1, wherein the heterocyclyl is selected from pyrazinyl, piperidinyl, morpholinyl, and pyrazolyl.
 10. The compound of claim 9, wherein the heterocyclyl is morpholinyl R³ is selected from —C(CH₃)₃, —NH-cyclopropyl, —CH₂-pyrazin-2-yl, -pyrazin-2-yl, —CH₂-morpholin-4-yl, and 5-methyl-1-H-pyrazol-4-yl.
 11. The compound of claim 1, wherein any alkyl, alkylene, heterocyclyl, and heteroaryl portion of R³ is optionally and independently substituted with one or more substituents selected from the group consisting of —OH, —SH, nitro, halogen, amino, cyano, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl or C₂-C₁₂ alkynyl group, C₁-C₁₂ alkoxy, C₁-C₁₂ haloalkyl, C₁-C₁₂ haloalkoxy and C₁-C₁₂ alkyl sulfanyl.
 12. The compound of claim 1, wherein any alkyl, alkylene, heterocyclyl, and heteroaryl portion of R³ is optionally and independently substituted with an amino group having the formula —N(R⁵)₂, wherein each R⁵ is independently selected from hydrogen and C₁-C₄ alkyl.
 13. The compound of claim 1, wherein: any heteroaryl portion of R³ is optionally and independently substituted with one or more substituents selected from the group consisting of —OH, —SH, nitro, halogen, amino, cyano, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, C₁-C₁₂ haloalkyl, C₁-C₁₂ haloalkoxy and C₁-C₁₂ alkyl sulfanyl; and any alkyl, alkylene or heterocyclyl portion of R³ is optionally and independently substituted with one or more substituents selected from the group consisting of oxo, —OH, —SH, nitro, halogen, amino, cyano, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, C₁-C₁₂ haloalkyl, C₁-C₁₂ haloalkoxy and C₁-C₁₂ alkyl sulfanyl.
 14. The compound of claim 1, wherein R³ is selected from —N(R⁴)—(C₃-C₆ cycloalkyl), —C₃-C₆ alkyl, —(C₀-C₁ alkylene)-heterocyclyl, and —(C₀-C₁ alkylene)-heteroaryl, wherein: any alkyl or alkylene portion of R³ is optionally substituted with —N(R⁵)₂, wherein each R⁵ is independently selected from hydrogen and C₁-C₄ alkyl; any heterocyclyl, and heteroaryl portion of R³ comprises at least one nitrogen atom in a ring; and any heterocyclyl, and heteroaryl portion of R³ is optionally substituted with C₁-C₄ alkyl.
 15. The compound of claim 14, wherein R³ is selected from —C(CH₃)₃, —CH(NH₂)—CH(CH₃)₂, —NH-cyclopropyl, —(CH₂)₀₋₁-pyrazinyl, piperidinyl, hydroxypiperidinyl, N-methylpiperidinyl, —CH₂-morpholin-4-yl, and methylpyrazolyl.
 16. The compound of claim 15, wherein R³ is selected from —C(CH₃)₃, —CH(NH₂)—CH(CH₃)₂, —NH-cyclopropyl, —(CH₂)₀₋₁-pyrazin-2-yl, piperidin-3-yl, —CH₂-morpholin-4-yl, and 5-methyl-1-H-pyrazol-4-yl.
 17. The compound of claim 16, wherein R³ is selected from —C(CH₃)₃, —NH-cyclopropyl, —CH₂-pyrazin-2-yl, -pyrazin-2-yl, —CH₂-morpholin-4-yl, and 5-methyl-1-H-pyrazol-4-yl.
 18. A compound represented by any any one of the structural formulas set forth below: Cmpd No. Compound Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

or a pharmaceutically acceptable salt thereof.
 19. (canceled)
 20. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 21. A method for treating a disorder associated with CRM1 activity, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to claim
 20. 22-31. (canceled) 